Agonists of guanylate cyclase and their uses

ABSTRACT

This invention provides a method of colonic cleansing.

RELATED APPLICATIONS

This application is a continuation of U.S. application No. 14/189,645,filed Feb. 25, 2014, which claims priority to, and benefit of, the U.S.Provisional Application No. 61/768,902, filed on Feb. 25, 2013, thecontents of which are incorporated herein by reference in theirentireties.

INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING

The contents of the text file named, which was created on Dec. 16, 2016and is 150 KB in size, are hereby incorporated by reference in theirentireties.

FIELD OF THE INVENTION

The present invention relates to colon cleansing compositions andmethods of use of such compositions.

BACKGROUND OF THE INVENTION

Colon cleansing is important prior to numerous diagnostic and surgicalprocedures, for example before colonoscopy, barium enema examination orcolon surgery. It is also useful for preventing infection after surgeryon the lower intestine. Colon cleansing is also known as colon clearing.

A variety of methods for colon cleansing are known. Dietarymanipulation, laxatives, cathartics and enemas were traditionally used(Thomas, G. et al., Gastroenterology, 1982, 82, 435 437). Sodiumphosphate solutions (Clarkston, W. K. et al., GastrointestinalEndoscopy, 1996, 43, 43 48) and magnesium citrate/sodium picosulphatesolutions (Regev, A. et al., Am. J. Gastroenterol., 1998, 93, 1478 1482)have also been used.

Those methods suffer from various drawbacks. Dietary manipulation andlaxatives are time consuming; enemas are unpleasant for the patient; anddangerous salt and water losses may occur with cathartics, enemas andwith sodium phosphate solutions.

Therefore, there is a great need to identify novel colon cleansingcompositions and methods without these drawbacks.

SUMMARY OF THE INVENTION

The present invention provides a method of colonic cleansing byadministering to a subject in need thereof an effective amount of aguanylate cyclase receptor agonist (GCRA) peptide or its derivative oranalog.

In some embodiments, the peptide is bicyclic GCRA peptide.

In some embodiments, the method further comprises administering to thesubject an effective amount of an osmotic colonic evacuant. Preferably,the osmotic colonic evacuant is magnesium citrate or a phosphate saltlaxative.

In some embodiment, the method further comprises administering to thesubject an effective amount of L-glucose, lubiprostone (Amitiza),prucalopride, an agent for treating chronic constipation, or anycombination thereof. The effective amount of L-glucose is a unit dose of20 g-200 g. Preferably, the GCRA peptide or its derivative or analog ofthe present invention is used in combination with an osmotic liquidprep, such as SUPREP® Bowel Prep Kit (sodium sulfate potassium sulfate,magnesium sulfate).

In some embodiment, the method further comprises administering to thesubject an effective amount of a cGMP-specific phosphodiesteraseinhibitor. The cGMP-specific phosphodiesterase inhibitor is selectedfrom the group consisting of sulindac sulfone, zaprinast, motapizone,vardenafil, and sildenafil.

The effective amount of a GCRA peptide is a unit dose of 0.01 mg to 60mg. Preferably, the effective amount of a GCRA peptide is a unit dose of6.0 mg.

The present invention provides a formulation that comprises a mixture of(1) a composition having an inert carrier coated with GCRA peptides andan enteric coating that releases the peptides at pH 5.0; and (2) acomposition having an inert carrier coated with GCRA peptides and anenteric coating that releases the peptides at pH 6.0 or pH 7.0.

In some embodiments, the inert carrier is a selected from mannitol,lactose, a microcrystalline cellulose, or starch.

In some embodiments, the amount of GCRA peptide per unit dose is from 1mg to 60 mg when the GCRA peptide is SP-304 (SEQ ID NO: 1) or SP-333(SEQ ID NO: 9) or their derivatives or analogs.

In some embodiments, the amount of GCRA peptide per unit dose is from0.3 mg to 3.0 mg when the GCRA peptide is an E. coli ST peptide,linaclotide derivative or analog.

The present invention further provides a method of colonic cleansing byadministering to a subject in need thereof any formulations describedherein.

In any methods or any formulations described herein, the GCRA peptideconsists essentially of the sequence of any one of SEQ ID NO: 1-346. Forexample, the peptide is bicyclic consisting essentially of the sequenceof any one of SEQ ID NO: 1-54, 99-241, and 253-346. Preferably, the GCRApeptide is SEQ ID NO: 1, 9, 55 or 56.

Other features and advantages of the invention will be apparent from andare encompassed by the following detailed description and claims.

DETAILED DESCRIPTION

The present invention is based upon the development of agonists ofguanylate cyclase-C (GC-C). The agonists are analogs of uroguanylin,guanylin, lymphoguanylin and ST peptides.

Physicians and surgeons have developed a variety of means to achieve thedesired level of colon cleansing. The use of dietary restrictions,laxatives, enemas, and whole-bowel lavage solutions, alone or incombinations has been employed.

However, these preparations have several drawbacks. For example, becausemost of commercially available solutions are isotonic, patients arerequired to ingest a significant amount of volume of these solutions, upto one eight ounce glass every ten minutes for a total of one gallon offluid, to achieve effective purging. Sodium sulfate and phosphate saltshave been used as laxatives. However, because of their small volumes,when used in this fashion they do not sufficiently clean the colon fordiagnostic or surgical procedures. Another drawback of thesepreparations is their unpleasant, bitter, saline taste. This can promotenausea and vomiting in sensitive patients—thereby preventing ingestion.

The gualylate cyclase-C agonists of the present invention provideunexpected and superior effect than previous means for colon cleansing.These agonists are very specific and are relatively more stable ingastrointestinal (GI) tract, because they have relatively higherresistance to degradation at the N-terminus and C-terminus fromcarboxypeptidases and/or by other proteolytic enzymes such as thosepresent in the stimulated human intestinal fluid (SIF). In addition,these agonists can significantly increase intestinal motility anddecrease water absorption. Therefore, they are excellent colon cleansingcompositions with high specificity, high efficiency, low volume/dosage,and no unpleasant taste, thus providing improved patients compliance.This is particularly true when the GCRA peptide or its derivative oranalog of the present invention or any composition/formulation describedherein is used in combination with a colon cleansing agent.

Preferably, the GCRA peptide or its derivative or analog of the presentinvention or any composition/formulation described herein is used incombination with L-glucose, cholera toxin, osmotic colonic evacuants,cathartic, laxatives, agents for treating chronic constipation and/or anosmotic liquid prep. For example, an osmotic liquid prep is SUPREP®Bowel Prep Kit (sodium sulfate potassium sulfate, magnesium sulfate).

Accordingly, the present invention provides compositions comprising atleast one GCRA peptide (i.e., GCC agonist peptide), at least one entericcoating which releases the peptide at a specific pH (e.g., pH 4.0, pH5.0, pH 6.0, or pH 7) and an inert carrier.

The present invention also provides a formulation comprising a mixtureof (1) a composition having an inert carrier coated with GCRA and anenteric coating that releases the peptides at pH 5.0 and (2) acomposition having an inert carrier coated with GCRA peptides and anenteric coating that releases the peptides at pH 6.0 or pH 7.0.

In some embodiments, the GCRA peptide is any one of SEQ ID NO: 1-346. Insome embodiments, the GCRA peptide is SEQ ID NO: 1, 9, 55 or 56. In someembodiments, the inert carrier is selected from the group consisting ofsorbitol, mannitol, EMDEX, and starch. In some embodiments, the carrieris mannitol (e.g., MANNOGEM) or microcrystalline cellulose (e.g.PROSOLV, CELPHERE®, CELPHERE® beads). In a preferred embodiment, thecarrier is microcrystalline cellulose sphere or sphericalmicrocrystalline cellulose, such as Celphere® SCP-100.

A composition may comprise an enteric coating which releases drug at pH5 and an inert carrier coated with GCRA peptides.

A composition may comprise an enteric coating which releases drug at pH6 and an inert carrier coated with GCRA peptides.

A composition may comprise an enteric coating which releases drug at pH7 and an inert carrier coated with GCRA peptides.

A composition may comprise an enteric coating which releases drug in apH range of 4.5 to 5.5 or in a pH range of 5.5 to 6.5 at duodenum orjejunum and an inert carrier coated with GCRA peptides.

A composition may comprise an enteric coating which releases drug in apH range of 5.5 to 6.5 or in a pH range of 6.5 to 7.5 at ileum, terminalileum, or ascending colon and an inert carrier coated with GCRApeptides.

A composition may comprise a mixture of enteric coatings which releasedrug at pH 5 and pH 6 or pH 7 and an inert carrier coated with GCRApeptides.

The present invention further provides compositions comprising a mixtureof compositions that contain different peptides and/or that release thepeptides at different pH levels, so that a specific composition can bereleased at a specific region of the GI tract (e.g., duodenum, jejunum,ileum, terminal ileum, or ascending colon) at a specific amount and timeto maximize the cleansing effect. Preferred pH for duodenum or jejunumrelease is pH 4.5-5.5 or pH 5.5-6.5. Preferred pH for ileum, terminalileum, or ascending colon release is pH 5.5-6.5 or pH 6.5-7.5.

The mixture may comprise at least 2, 3, 4 or more compositions thatrelease the peptides at different pH levels (e.g., pH 5, pH 6, or pH 7).The mixture may comprise at least 2, 3, 4 or more compositions thatcontain different GCRA peptides. A skilled artisan can determine theratio of these compositions within the mixture, for example, accordingto the activity of each peptide, solubility of each peptide, and/or thetargeting region of the GI tract.

The present invention also provides methods for colonic cleansing byadministering to a subject in need thereof an effective amount of a GCRApeptide.

The GCRA peptides (i.e., gualylate cyclase-C agonists) according to theinvention include amino acid sequences represented by Formulae I-XXI,their corresponding α-aminoadipic acid (Aad) derivatives (e.g., FormulaeI-Aad, II-Aad, III-Aad, IV-Aad, V-Aad, VI-Aad, VII-a-Aad, VII-b-Aad,VIII-Aad, IX-Aad, XVIII-Aad or XXI-Aad), as well as those amino acidsequence summarized below in Tables 1-8. The gualylate cyclase-Cagonists according to the invention are collectively referred to hereinas “GCRA peptides”.

TABLE 1 GCRA Peptides (SP-304 and Derivatives) SEQ Position of ID NameDisulfide bonds Structure NO SP-304 C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶  1 SP-326 C3:C11, C6:C14Asp¹-Glu²-Cys³-Glu⁴-Leu⁵-Cys⁶-Val⁷-Asn⁸-Val⁹-Ala¹⁰-Cys¹¹-Thr¹²-Gly¹³-Cys¹⁴-Leu¹⁵  2 SP-327 C3:C11, C6:C14Asp¹-Glu²-Cys³-Glu⁴-Leu⁵-Cys⁶-Val⁷-Asn⁸-Val⁹-Ala¹⁰-Cys¹¹-Thr¹²-Gly¹³-Cys¹⁴  3 SP-328 C2:C10, C5:C13Glu¹-Cys²-Glu³-Leu⁴-Cys⁵-Val⁶-Asn⁷-Val⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³-Leu¹⁴  4 SP-329 C2:C10, C5:C13Glu¹-Cys²-Glu³-Leu⁴-Cys⁵-Val⁶-Asn⁷-Val⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³   5SP-330 C1:C9, C4:C12Cys¹-Glu²-Leu³-Cys⁴-Val⁵-Asn⁶-Val⁷-Ala⁸-Cys⁹-Thr¹⁰-Gly¹¹-Cys¹²-Leu¹³   6SP-331 C1:C9, C4:C12Cys¹-Glu²-Leu³-Cys⁴-Val⁵-Asn⁶-Val⁷-Ala⁸-Cys⁹-Thr¹⁰-Gly¹¹-Cys¹²   7 SP332C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶  8 SP-333 C4:C12, C7:C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶  9 SP-334 C4:C12, C7:C15dAsn¹-dAsp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶ 10 SP-335 C4:C12, C7:C15dAsn¹-dAsp²-dGlu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶ 11 SP-336 C4:C12, C7:C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶ 12 SP-337 C4:C12, C7:C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-dLeu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶ 13 SP-338 C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵ 14 SP-342 C4:C12, C7:C15PEG3-Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶-PEG3 15 SP-343 C4:C12, C7:C15PEG3-dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶-PEG3 16 SP-344 C4:C12, C7:C15PEG3-dAsn¹-dAsp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶-PEG3 17 SP-347 C4:C12, C7:C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶-PEG3 18 SP-348 C4:C12, C7:C15PEG3-Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶ 19 SP-350 C4:C12, C7:C15PEG3-dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶ 20 SP-352 C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶-PEG3 21 SP-358 C4:C12, C7:C15PEG3-dAsn¹-dAsp²-dGlu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶- 22 PEG3 SP-359 C4:C12, C7:C15PEG3-dAsn¹-dAsp²-dGlu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn₉-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶ 23 SP-360 C4:C12, C7:C15dAsn¹-dAsp²-dGlu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys1²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶-PEG3 24 SP-361 C4:C12, C7:C15dAsn¹-dAsp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-G1y¹⁴-Cys¹⁵-dLeu¹⁶-PEG3 25 SP-362 C4:C12, C7:C15PEG3-dAsn¹-dAsp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶ 26 SP-368 C4:C12, C7:C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dNal¹⁶ 27 SP-369 C4:C12, C7:C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷⁻AIB⁸-Asn⁹-AIB¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶ 28 SP-370 C4:C12, C7:C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Asp[Lactam]⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Orn¹⁵-dLeu¹ 29 SP-371 C4:C12, C7:C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶ 30 SP-372 C4:C12, C7:C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Ser⁶-Cys7⁻Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶ 31 N1 C4:C12, C7:C15PEG3-dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶⁻PEG3 32 N2 C4:C12, C7:C15PEG3-dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶ 33 N3 C4:C12, C7:C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶-PEG3 34 N4 C4:C11, C7:C15PEG3-dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Ser⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶-PEG3 35 N5 C4:C12, C7:C15PEG3-dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Ser⁶-Cys⁷-Val₈-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶ 36 N6 C4:C12, C7:C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Ser⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶-PEG3 37 N7 C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶ 38 N8 C4:C12, C7:C15PEG3-Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶-PEG3 39 N9 C4:C12, C7:C15PEG3-Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶ 40 N10 C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶-PEG3 41 N11 C4:C12, C7:C15PEG3-Asn¹-Asp²-Glu³-Cys4-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dSer¹⁶-PEG3 42 N12 C4:C12, C7:C15PEG3-Asn¹-Asp²-Glu³-Cys4-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dSer¹⁶ 43 N13 C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dSer¹⁶-PEG3 44 Formula C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Xaa⁵-Xaa⁶-Cys⁷-Xaa⁸-Xaa⁹-Xaa¹⁰-Xaa¹¹-Cys¹²-Xaa¹³-Xaa¹⁴-Cys¹⁵-Xaa¹⁶ 45 I Formula C4:C12, C7:C15Xaa_(n1)-Cys⁴-Xaa⁵-Xaa⁶-Cys⁷-Xaa⁸-Xaa⁹-Xaa¹⁰-Xaa¹¹-Cys¹²-Xaa¹³-Xaa¹⁴-Cys¹⁵-Xaa_(n2)¹⁶  46 II Formula C4:C12, C7:C15Xaa_(n1)-Maa⁴-Glu⁵-Xaa⁶-Maa⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Maa¹²-Thr¹³-Gly¹⁴-Maa¹⁵-Xaa_(n2) 47 III Formula C4:C12, C7:C15Xaa_(n1)-Maa⁴-Xaa⁵-Xaa⁶-Maa⁷-Xaa⁸-Xaa⁹-Xaa¹⁰-Xaa¹¹-Maa¹²-Xaa¹³-Xaa¹⁴-Maa¹⁵-Xaa_(n2) 48 IV Formula C4:C12, C7:C15Asn¹-Asp²-Asp³-Cys⁴-Xaa⁵-Xaa⁶-Cys⁷-Xaa⁸-Asn⁹-Xaa¹⁰-Xaa¹¹-Cys¹²-Xaa¹³-Xaa¹⁴-Cys¹⁵-Xaa¹⁶ 49 V Formula C4:C12, C7:C15dAsn¹-Glu²-Glu³-Cys⁴-Xaa⁵-Xaa⁶-Cys⁷-Xaa⁸-Asn⁹-Xaa¹⁰-Xaa¹¹-Cys¹²-Xaa¹³-Xaa¹⁴-Cys¹⁴-d-Xaa¹⁶ 50 VI Formula C4:C12, C7:C15dAsn¹-dGlu²-Asp³-Cys⁴-Xaa⁵-Xaa⁶-Cys⁷-Xaa⁸-Asn⁹-Xaa¹⁰-Xaa¹¹-Cvs¹²-Xaa¹³-Xaa¹⁴-Cys¹⁵-d-Xaa¹⁶ 51 VII-a Formula C4:C12, C7:C15dAsn¹-dAsp²-Glu³-Cys⁴-Xaa⁵-Xaa⁶-Cys⁷-Xaa⁸-Asn⁹-Xaa¹⁰-Xaa¹¹-Cvs¹²-Xaa¹³-Xaa¹⁴-Cys¹⁵-d-Xaa¹⁶ 52 VII-b Formula C4:C12, C7:C15dAsn¹-dAsp²-dGlu³-Cys⁴-Xaa⁵-Xaa⁶-Cys⁷-Xaa⁸-Tyr9-Xaa¹⁰-Xaa¹¹-Cys¹²-Xaa¹³-Xaa¹⁴-Cys¹⁵-d-Xaa¹⁶ 53 VIII Formula C4:C12, C7:C15dAsn¹-dGlu²-dGlu³-Cys⁴-Xaa⁵-Xaa⁶-Cys⁷-Xaa⁸-Tyr⁹-Xaa¹⁰-Xaa¹¹-Cys¹²-Xaa¹³-Xaa¹⁴-Cys¹⁵-d-Xaa¹⁶ 54 IX Formula C4:C12, C7:C15Xaa_(n1)-Cys⁴-Xaa⁵-Xaa⁶-Xaa⁷-Xaa⁸-Xaa⁹-Xaa¹⁰-Xaa¹¹-Cys¹²-Xaa¹³-Xaa¹⁴-Xaa¹⁵-Xaa_(n2)¹⁶ 250 XXI

TABLE 2 Linaclotide and Derivatives Position of SEQ ID NameDisulfide Bonds Structure NO: SP- C1:C6, C2:C10, C5:C13Cys¹-Cys²-Glu³-Tyr⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³-Tyr¹⁴55 339 (linaclotide) SP-340 C1:C6, C2:C10, C5:C13Cys¹-Cys²-Glu³-Tyr⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³ 56SP-349 C1:C6, C2:C10, C5:C13PEG3-Cys¹-Cys²-Glu³-Tyr⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³-Tyr¹⁴-57 PEG3 SP-353 C3:C8, C4:C12, C7:C15Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Ser⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-58 Tyr¹⁶ SP-354 C3:C8, C4:C12, C7:C15Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Phe⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-59 Tyr¹⁶ SP-355 C1:C6, C2:C10, C5:C13Cys¹-Cys²-Glu³ Tyr⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³-dTyr¹⁴60 SP-357 C1:C6, C2:C10, C5:C13PEG3-Cys¹-Cys²-Glu³-Tyr⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³-Tyr¹⁴61 SP-374 C3:C8, C4:C12, C7:C15Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-62 Tyr¹⁶ SP-375 C3:C8, C4:C12, C7:C15Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Ser⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-63 dTyr16 SP-376 C3:C8, C4:C12, C7:C15dAsn¹-Phe²-Cys³-Cys⁴-Glu⁵-Ser⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-64 Tyr¹⁶ SP-377 C3:C8, C4:C12, C7:C15dAsn¹-Phe²-Cys³-Cys⁴-Glu⁵-Ser⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-65 dTyr¹⁶ SP-378 C3:C8, C4:C12, C7:C15Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-66 dTyr¹⁶ SP-379 C3:C8, C4:C12, C7:C15dAsn¹-Phe²-Cys³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-67 Tyr¹⁶ SP-380 C3:C8, C4:C12, C7:C15dAsn¹-Phe²-Cys³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-68 dTyr¹⁶ SP-381 C3:C8, C4:C12, C7:15Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Phe⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-69 dTyr¹⁶ SP-382 C3:C8, C4:C12, C7:15dAsn¹-Phe²-Cys³-Cys⁴-Glu⁵-Phe⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-70 Tyr¹⁶ SP-383 C3:C8, C4:C12, C7:15dAsn¹-Phe²-Cys³-Cys⁴-Glu⁵-Phe⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-71 dTyr¹⁶ SP384 C1:C6, C2:C10, C5:C13Cys¹-Cys²-Glu³-Tyr⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³-Tyr¹⁴-PEG372 N14 C1:C6, C2:C10, C5:C13PEG3-Cys¹-Cys²-Glu³-Tyr⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³-PEG373 N15 C1:C6, C2:C10, C5:C13PEG3-Cys¹-Cys²-Glu³-Tyr⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³74 N16 C1:C6, C2:C10, C5:C13Cys¹-Cys²-Glu³-Tyr⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³-PEG375 N17 C3:C8, C4:C12, C7:C15PEG3-Asn¹-Phe²-Cys³-Cys⁴-Gly⁵-Ser⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴76 Cys¹⁵-Tyr¹⁶-PEG3 N18 C3:C8, C4:C12, C7:C15PEG3-Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Ser⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-77 Cys¹⁵-Tyr¹⁶ N19 C3:C8, C4:C12, C7:C15Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Ser⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-78 Tyr¹⁶-PEG3 N20 C3:C8, C4:C12, C7:C15PEG3-Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Phe⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-79 Cys¹⁵-Tyr¹⁶-PEG3 N21 C3:C8, C4:C12, C7:C15PEG3-Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Phe⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-80 Cys¹⁵-Tyr¹⁶ N22 C3:C8, C4:C12, C7:C15Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Phe⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-G1y¹⁴-Cys¹⁵-81 Tyr¹⁶-PEG3 N23 C3:C8, C4:C12, C7:C15PEG3-Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-82 Cys¹⁵-Tyr¹⁶-PEG3 N24 C3:C8, C4:C12, C7:C15PEG3-Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-83 Cys¹⁵-Tyr¹⁶ N25 C3:C8, C4:C12, C7:C15Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-84 Tyr¹⁶-PEG3 N26 C1:C6, C2:C10, C5:C13Cys¹-Cys²-Glu³-Ser⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³-Tyr¹⁴85 N27 C1:C6, C2:C10, C5:C13Cys¹-Cys²-Glu³-Phe⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr^(1l)-Gly¹²-Cys¹³-Tyr¹⁴86 N28 C1:C6, C2:C10, C5:C13Cys¹-Cys²-Glu³-Ser⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³- 87N29 C1:C6, C2:C10, C5:C13Cys¹-Cys²-Glu³-Phe⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³ 88N30 1:6, 2:10, 5:13Pen¹-Pen²-Glu³-Tyr⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³ 89Tyr¹⁴ N31 1:6, 2:10, 5:13Pen¹-Pen²-Glu³-Tyr⁴-Pen⁵-Pen⁶-Asn⁷-Pro⁸-Ala⁹-Pen¹⁰-Thr¹¹-Gly¹²-Pen¹³ 90Formula X C9:C14, C10:C18,Xaa¹-Xaa²-Xaa³-Xaa⁴-Xaa⁵-Xaa⁶-Asn⁷-Tyr⁸-Cys⁹-Cys¹⁰-Xaa¹¹-Tyr¹¹-Cys¹³-Cys¹⁴-Xaa¹⁵91 C13:C21 Xaa¹⁶-Xaa¹⁷-Cys¹⁸-Xaa¹⁹-Xaa²⁰-Cys²¹-Xaa²² Formula XIC9:C14, C10:C18,Xaa¹-Xaa²-Xaa³-Xaa⁴-Xaa⁵-Xaa⁶-Asn⁷-Phe⁸-Cys⁹-Cys¹⁰-Xaa¹¹-Phe¹²-Cys¹³-Cys¹⁴-Xaa¹⁵92 C13:C21 Xaa¹⁶-Xaa¹⁷-Cys¹⁸-Xaa¹⁹-Xaa²⁰-Cys²¹-Xaa²² Formula XIIC3:C8, C4:C12, C7:C15Asn¹-Phe²-Cys³-Cys⁴-Xaa⁵-Phe⁶-Cys⁷-Cys⁸-Xaa⁹-Xaa¹⁰-Xaa¹¹-Cys¹²-Xaa¹³-Xaa¹⁴-Cys¹⁵-93 Xaa¹⁶ Formula XIII 3:8, 4:12, 7:15Asn¹-Phe²-Pen³-Cys⁴-Xaa⁵-Phe⁶-Cys⁷-Pen⁸-Xaa⁹-Xaa¹⁰-Xaa¹¹-Cys¹²-Xaa¹³-Xaa¹⁴-Cys¹⁵-94 Xaa¹⁶ Formula XIV 3:8, 4:12, 7:15Asn¹-Phe²-Maa³-Maa⁴-Xaa⁵-Xaa⁶-Maa⁷-Maa⁸-Xaa⁹-Xaa¹⁰-Xaa¹¹-Maa¹²-Xaa¹³-Xaa¹⁴-95 Maa¹⁵-Xaa¹⁶ Formula XV 1:6, 2:10, 5:13Maa¹-Maa²-Glu³-Xaa⁴-Maa⁵-Maa⁶-Asn⁷-Pro⁸-Ala⁹-Maa¹⁰-Thr¹¹-Gly¹²-Maa¹³-Tyr¹⁴96 Formula XVI 1:6, 2:10, 5:13Maa¹-Maa²-Glu³-Xaa⁴-Maa⁵-Maa⁶-Asn⁷-Pro⁸-Ala⁹-Maa¹⁰-Thr¹¹-Gly¹²-Maa¹³ 97Formula XVII 1:6, 2:10, 5:13Xaa_(n3)-Maa¹-Maa²-Xaa³-Xaa⁴-Maa⁵-Maa⁶-Xaa⁷-Xaa⁸-Xaa⁹-Maa¹⁰-Xaa¹¹-Xaa¹²-Maa¹³-98 Xaa_(n2)

TABLE 3 GCRA Peptides Position of SEQ Disulfide ID Name bonds StructureNO: SP-363 C4:C12, C7:C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-  99Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu-AMIDE¹⁶ SP-364 C4:C12, C7:C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹- 100Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dSer¹⁶ SP-365 C4:C12, C7:C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹- 101Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dSer-AMIDE¹⁶ SP-366 C4:C12, C7:C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹- 102Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dTyr¹⁶ SP-367 C4:C12, C7:C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰⁻Ala¹¹- 103Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dTyr-AMIDE¹⁶ SP-373 C4:C12, C7:C15Pyglu¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹- 104Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu-AMIDE¹⁶ / C4:C12, C7:C15Pyglu¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹- 251Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶ SP- C4:C12, C7:C15PEG3-Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰- 105 304diPEGAla¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶-PEG3 SP-304N- C4:C12, C7:C15PEG3-Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰- 106 PEGAla¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶ SP-304C- C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵⁻Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹- 107 PEGCys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶-PEG3

TABLE 4 SP-304 Analogs, Uroguanylin, and Uroguanylin Analogs Position ofSEQ ID Name Disulfide bonds Structure NO Formula C4:C12, C7:C15Xaa¹-Xaa²-Xaa³-Maa⁴-Xaa⁵-Xaa⁶-Maa⁷-Xaa⁸-Xaa⁹-Xaa¹⁰-Xaa¹¹-Maa¹²-Xaa¹³-Xaa¹⁴-Maa¹⁵-Xaa¹⁶108 XVIII Uroguanylin C4:C12, C7:C15Asn¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶109 N32 C4:C12, C7:C15Glu¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶110 N33 C4:C12, C7:C15Glu¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶111 N34 C4:C12, C7:C15Glu¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶112 N35 C4:C12, C7:C15Glu¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶113 N36 C4:C12, C7:C15Asp¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶114 N37 C4:C12, C7:C15Asp¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶115 N38 C4:C12, C7:C15Asp¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶116 N39 C4:C12, C7:C15Asp¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶117 N40 C4:C12, C7:C15Gln¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶118 N41 C4:C12, C7:C15Gln¹-Asp²-Glu³-Cvs⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶119 N42 C4:C12, C7:C15Gln¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶120 N43 C4:C12, C7:C15Gln¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶121 N44 C4:C12, C7:C15Lys¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶122 N45 C4:C12, C7:C15Lys¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶123 N46 C4:C12, C7:C15Lys¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶124 N47 C4:C12, C7:C15Lys¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶125 N48 C4:C12, C7:C15Glu¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶126 N49 C4:C12, C7:C15Glu¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶127 N50 C4:C12, C7:C15Glu¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶128 N51 C4:C12, C7:C15Glu¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶129 N52 C4:C12, C7:C15Asp¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶130 N53 C4:C12, C7:C15Asp¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶131 N54 C4:C12, C7:C15Asp¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶132 N55 C4:C12, C7:C15Asp¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶133 N56 C4:C12, C7:C15Gln¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶134 N57 C4:C12, C7:C15Gln¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶135 N58 C4:C12, C7:C15Gln¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶136 N59 C4:C12, C7:C15Gln¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶137 N60 C4:C12, C7:C15Lys¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶138 N61 C4:C12, C7:C15Lys¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶139 N62 C4:C12, C7:C15Lys¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶140 N63 C4:C12, C7:C15Lys¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶141 N65 C4:C12, C7:C15Glu¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶142 N66 C4:C12, C7:C15Glu¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶143 N67 C4:C12, C7:C15Glu¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶144 N68 C4:C12, C7:C15Glu¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶145 N69 C4:C12, C7:C15Asp¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶146 N70 C4:C12, C7:C15Asp¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶147 N71 C4:C12, C7:C15Asp¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶148 N72 C4:C12, C7:C15Asp¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶149 N73 C4:C12, C7:C15Gln¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶150 N74 C4:C12, C7:C15Gln¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶151 N75 C4:C12, C7:C15Gln¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶152 N76 C4:C12, C7:C15Gln¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶153 N77 C4:C12, C7:C15Lys¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶154 N78 C4:C12, C7:C15Lys¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶155 N79 C4:C12, C7:C15Lys¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶156 N80 C4:C12, C7:C15Lys¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶157 N81 C4:C12, C7:C15Glu¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶158 N82 C4:C12, C7:C15Glu¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶159 N83 C4:C12, C7:C15Glu¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶160 N84 C4:C12, C7:C15Glu¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶161 N85 C4:C12, C7:C15Asp¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶162 N86 C4:C12, C7:C15Asp¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶163 N87 C4:C12, C7:C15Asp¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶164 N88 C4:C12, C7:C15Asp¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶165 N89 C4:C12, C7:C15Gln¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶166 N90 C4:C12, C7:C15Gln¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶167 N91 C4:C12, C7:C15Gln¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶168 N92 C4:C12, C7:C15Gln¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶169 N93 C4:C12, C7:C15Lys¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶170 N94 C4:C12, C7:C15Lys¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶171 N95 C4:C12, C7:C15Lys¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶172 N96 C4:C12, C7:C15Lys¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶173

TABLE 5 Guanylin and Analogs Position of SEQ Disulfide ID  Name bondsStructure NO Formula 4:12,7:15Xaa¹-Xaa²-Xaa³-Maa⁴-Xaa⁵-Xaa⁶-Maa⁷-Xaa⁸-Xaa⁹-Xaa¹⁰-Xaa¹¹-Maa¹²-Xaa¹³-Xaa¹⁴-Maa¹⁵174 XIX Guanylin C4:C12,Ser¹-His²-Thr³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ala⁸-Phe⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵175 C7:C15 Guanylin C4:C12,Pro¹-Gly²-Thr³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ala⁸-Tyr⁹-Ala¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵252 C7:C15 Human C4:C12,Pro¹-Gly²-Thr³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ala⁸-Tyr⁹-Ala¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵Guanylin C7:C15 N97 C4:C12,Ser¹-His²-Thr³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵176 C7:C15 N98 C4:C12,Ser¹-His²-Thr³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵177 C7:C15 N99 C4:C12,Ser¹-His²-Thr³-Cys⁴-Glu⁵-Val⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵178 C7:C15 N100 C4:C12,Ser¹-His²-Thr³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵179 C7:C15 N101 C4:C12,Ser¹-His²-Thr³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵180 C7:C15 N102 C4:C12,Ser¹-His²-Thr³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵181 C7:C15 N103 C4:C12,Ser¹-His²-Thr³-Cys⁴-Glu⁵-Val⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵182 C7:C15 N104 C4:C12,Ser¹-His²-Thr³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵183 C7:C15 N105 C4:C12,Ser¹-His²-Thr³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵184 C7:C15 N106 C4:C12,Ser¹-His²-Thr³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵185 C7:C15 N107 C4:C12,Ser¹-His²-Thr³-Cys⁴-Glu⁵-Val⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵186 C7:C15 N108 C4:C12,Ser¹-His²-Thr³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵187 C7:C15 N109 C4:C12,Ser¹-His²-Thr³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵188 C7:C15 N110 C4:C12,Ser¹-His²-Thr³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵189 C7:C15 N111 C4:C12,Ser¹-His²-Thr³-Cys⁴-Glu⁵-Val⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵190 C7:C15 N112 C4:C12,Ser¹-His²-Thr³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵191 C7:C15 N113 C4:C12,Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵192 C7:C15 N114 C4:C12,Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵193 C7:C15 N115 C4:C12,Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Val⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵194 C7:C15 N116 C4:C12,Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵195 C7:C15 N117 C4:C12,Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵196 C7:C15 N118 C4:C12,Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵197 C7:C15 N119 C4:C12,Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Val⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵198 C7:C15 N120 C4:C12,Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵199 C7:C15 N121 C4:C12,Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵200 C7:C15 N122 C4:C12,Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵201 C7:C15 N123 C4:C12,Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Val⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵202 C7:C15 N124 C4:C12,Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵203 C7:C15 N125 C4:C12,Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵204 C7:C15 N126 C4:C12,Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵205 C7:C15 N127 C4:C12,Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Val⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵206 C7:C15 N128 C4:C12,Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵207 C7:C15

TABLE 6 Lymphoguanylin and Analogs Position of SEQ  Disulfide ID Namebonds Structure NO Formula 4:12Xaa¹-Xaa²-Xaa³-Maa⁴-Xaa⁵-Xaa⁶-Maa⁷-Xaa⁸-Xaa⁹-Xaa¹⁰-Xaa¹¹-Maa¹²-Xaa¹³-Xaa¹⁴-Xaa_(n1)¹⁵ 208 XX Lympho- C4:C12Gln¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly₁₄-Tyr¹⁵209 guanylin N129 C4:C12Gln¹-Glu²-Glu³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly₁₄-Tyr¹⁵210 N130 C4:C12Gln¹-Asp²-Glu³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly₁₄-Tyr¹⁵211 N131 C4:C12Gln¹-Asp²-Asp³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly₁₄-Tyr¹⁵212 N132 C4:C12Gln¹-Glu²-Asp³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly₁₄-Tyr¹⁵213 N133 C4:C12Gln¹-Glu²-Glu³-Cys⁴-Glu⁵-Glu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly₁₄-Tyr¹⁵214 N134 C4:C12Gln¹-Asp²-Glu³-Cys⁴-Glu⁵-Glu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly₁₄-Tyr¹⁵215 N135 C4:C12Gln¹-Asp²-Asp³-Cys⁴-Glu⁵-Glu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly₁₄-Tyr¹⁵216 N136 C4:C12Gln¹-Glu²-Asp³-Cys⁴-Glu⁵-Glu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly₁₄-Tyr¹⁵217 N137 C4:C12Gln¹-Glu²-Glu³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly₁₄-Tyr¹⁵218 N138 C4:C12Gln¹-Asp²-Glu³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly₁₄-Tyr¹⁵219 N139 C4:C12Gln¹-Asp²-Asp³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly₁₄-Tyr¹⁵220 N140 C4:C12Gln¹-Glu²-Asp³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly₁₄-Tyr¹⁵221 N141 C4:C12Gln¹-Glu²-Glu³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly₁₄-Tyr¹⁵222 N142 C4:C12Gln¹-Asp²-Glu³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly₁₄-Tyr¹⁵223 N143 C4:C12Gln¹-Asp²-Asp³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly₁₄-Tyr¹⁵224 N144 C4:C12Gln¹-Glu²-Asp³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly₁₄-Tyr¹⁵225 N145 C4:C12,Gln¹-Glu²-Glu³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-226 C7:C15 Ser¹⁶ N146 C4:C12,Gln¹-Asp²-Glu³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-227 C7:C15 Ser¹⁶ N147 C4:C12,Gln¹-Asp²-Asp³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-228 C7:C15 Ser¹⁶ N148 C4:C12,Gln¹-Glu²-Asp³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-229 C7:C15 Ser¹⁶ N149 C4:C12,Gln¹-Glu²-Glu³-Cys⁴-Glu⁵-Glu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-230 C7:C15 Ser¹⁶ N150 C4:C12,Gln¹-Asp²-Glu³-Cys⁴-Glu⁵-Glu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-231 C7:C15 Ser¹⁶ N151 C4:C12,Gln¹-Asp²-Asp³-Cys⁴-Glu⁵-Glu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-232 C7:C15 Ser¹⁶ N152 C4:C12,Gln¹-Glu²-Asp³-Cys⁴-Glu⁵-Glu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-233 C7:C15 Ser¹⁶ N153 C4:12,Gln¹-Glu²-Glu³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-234 C7:C15 Ser¹⁶ N154 C4:C12,Gln¹-Asp²-Glu³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-235 C7:C15 Ser¹⁶ N155 C4:C12,Gln¹-Asp²-Asp³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-236 C7:C15 Ser¹⁶ N156 C4:C12,Gln¹-Glu²-Asp³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-237 C7:C15 Ser¹⁶ N157 C4:C12,Gln¹-Glu²-Glu³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-238 C7:C15 Ser¹⁶ N158 C4:C12,Gln¹-Asp²-Glu³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-239 C7:C15 Ser¹⁶ N159 C4:C12,Gln¹-Asp²-Asp³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-240 C7:C15 Ser¹⁶ N160 C4:C12,Gln¹-Glu²-Asp³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-241 C7:C15 Ser¹⁶

TABLE 7 ST Peptide and Analogues Position of SEQ Disulfide ID Name bondsStructure NO STPeptide C9:14,Asn¹-Ser²-Ser³-Asn⁴-Ser⁵-Ser⁶-Asn⁷-Tyr⁸-Cys⁹-Cys¹⁰-Glu¹¹-Lys¹²-Cys¹³-242 C10:C18, Cys¹⁴-Asn¹⁵-Pro¹⁶-Ala¹⁷-Cys¹⁸-Thr¹⁹-Gly²⁰-Cys²¹-Tyr²²C13:C21 N161 C3:C8,PEG3-Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²- 243C4:C12, Thr¹³-Gly¹⁴-Cys¹⁵-Tyr¹⁶-PEG3 C7:C15 N162 C3:C8,PEG3-Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²- 244C4:C12, Thr¹³-Gly¹⁴-Cys¹⁵-Tyr¹⁶ C7:C15 N163 C3:C8,Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-245 C4:C12, Gly¹⁴-Cys¹⁵-Tyr¹⁶-PEG3 C7:C15 N164 C3:C8,Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-246 C4:C12, Gly¹⁴-Cys¹⁵-Tyr¹⁶ C7:15 N165 C3:C8,dAsn¹-Phe²-Cys³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²- 247C4:C12, Thr¹³-Gly¹⁴-Cys¹⁵-dTyr¹⁶ C7:C15 N166 C3:C8,Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-248 C4:C12, Gly¹⁴-Cys¹⁵-dTyr¹⁶ C7:15 N167 C3:C8,dAsn¹-Phe²-Cys³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²- 249C4:12, Thr¹³-Gly¹⁴-Cys¹⁵-Tyr¹⁶ C7:15

TABLE 8 Alpha-aminoadipic acid derivatives of GCRA Peptides Position ofSEQ Corres- Disulfide ID ponds to: bond Structure NO SP-304C4:C12, C7:C15Asn¹-Asp²-Aad³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶253 SP-326 C3:C11, C6:C14Asp¹-Aad²-Cys³-Glu⁴-Leu⁵-Cys⁶-Val⁷-Asn⁸-Val⁹-Ala¹⁰-Cys¹¹-Thr¹²-Gly¹³-Cys¹⁴-Leu¹⁵254 SP-327 C3:C11, C6:C14Asp¹-Aad²-Cys³-Glu⁴-Leu⁵-Cys⁶-Val⁷-Asn⁸-Val⁹-Ala¹⁰-Cys¹¹-Thr¹²-Gly¹³-Cys¹⁴255 SP-328 C2:C10, C5:C13Aad¹-Cys²-Glu³-Leu⁴-Cys⁵-Val⁶-Asn⁷-Val⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³-Leu¹⁴256 SP-329 C2:C10, C5:C13Aad¹-Cys²-Glu³-Leu⁴-Cys⁵-Val⁶-Asn⁷-Val⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³ 257SP332 C4:C12, C7:C15Asn¹-Asp²-Aad³-Cys⁴-Glu⁵⁻Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thru¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶258 SP-333 C4:C11, C7:C15dAsn¹-Asp²-Aad³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶259 SP-334 C4:C12, C7:C15dAsn¹-dAsp²-Aad³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶260 SP-336 C4:C12, C7:C15dAsn¹-Asp²-Aad³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶261 SP-337 C4:C12, C7:C15dAsn¹-Asp²-Aad³-Cys⁴-Glu⁵-dLeu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶262 SP-338 C4:C12, C7:C15Asn¹-Asp²-Aad³-Cys⁴-Glu⁵⁻Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵263 SP-342 C4:C12, C7:C15PEG3-Asn¹-Asp²-Aad³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶-PEG3264 SP-343 C4:C12, C7:C15PEG3-dAsn¹-Asp²-Aad³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶-PEG3265 SP-344 C4:C12, C7:C15PEG3-dAsn¹-dAsp²-Aad³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶-266 PEG3 SP-347 C4:C12, C7:C15dAsn¹-Asp²-Aad³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶-PEG3267 SP-348 C4:C12, C7:C15PEG3-Asn¹-Asp²-Aad³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶268 SP-350 C4:C12, C7:C15PEG3-dAsn¹-Asp²-Aad³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶269 SP-352 C4:C12, C7:C15Asn¹-Asp²-Aad³-Cys⁴-Glu⁵-Leu⁶-Cys7-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cysis-dLeu¹⁶-PEG3270 SP-359 C4:C11, C7:C15PEG3-dAsn¹-dAsp²-Aad³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶271 SP-360 C4:C12, C7:C15dAsn¹-dAsp²-Aad³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶-PEG3272 SP-368 C4:C12, C7:C15dAsn¹-Asp²-Aad³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dNal¹⁶273 SP-369 C4:C12, C7:C15dAsn¹-Asp²-Aad³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-AIB⁸-Asn⁹-AIB¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶274 SP-370 C4:C12, C7:C15dAsn¹-Asp²-Aad³-Cys⁴-Glu⁵-Leu⁶-Asp[Lactam]⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Orn¹⁵-dLeu¹⁶275 SP-371 C4:C12, C7:C15dAsn¹-Asp²-Aad³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶276 SP-372 C4:C12, C7:C15dAsn¹-Asp²-Aad³-Cys⁴-Glu⁵-Ser⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶277 N1 C4:C12, C7:C15PEG3-dAsn¹-Asp²-Aad³-Cys⁴-Glu⁵-Tyr6-Cys7-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶-PEG3278 N2 C4:C12, C7:C15PEG3-dAsn¹-Asp²-Aad³-Cys⁴-Glu⁵-Tyr⁶-Cys7-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶279 N3 C4:C12, C7:C15dAsn¹-Asp²-Aad³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Val⁸-Asn9-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶-PEG3280 N4 C4:C12, C7:C15PEG3-dAsn¹-Asp²-Aad³-Cys⁴-Glu⁵-Ser⁶-Cys7-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶-PEG3281 N5 C4:C12, C7:C15PEG3-dAsn¹-Asp²-Aad³-Cys⁴-Glu⁵-Ser⁶-Cys7-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶282 N6 C4:C11, C7:C15dAsn¹-Asp²-Aad³-Cys⁴-Glu⁵-Ser⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶-PEG3283 N7 C4:C12, C7:C15Asn¹-Asp²-Aad³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶284 N8 C4:C12, C7:C15PEG3-Asn¹-Asp²-Aad³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶-PEG3285 N9 C4:C12, C7:C15PEG3-Asn¹-Asp²-Aad³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶286 N10 C4:C12, C7:C15Asn¹-Asp²-Aad³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶-PEG3287 N11 C4:C12, C7:C15PEG3-Asn¹-Asp²-Aad³-Cys4-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dSer¹⁶-PEG3288 N12 C4:C12, C7:C15PEG3-Asn¹-Asp²-Aad³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dSer¹⁶289 N13 C4:C12, C7:C15Asn¹-Asp²-Aad-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dSer¹⁶-PEG3290 Formula I C4:C12, C7:C15Asn¹-Asp²-Aad³-Cys⁴-Xaa⁵-Xaa⁶-Cys⁷-Xaa⁸-Xaa⁹-Xaa¹⁰-Xaa¹¹-Cys¹²-Xaa¹³-Xaa¹⁴-Cys¹⁵-Xaa¹⁶291 (I-Aad) Formula II C4:C12,C7:C15Xa_(n1)-Cys⁴-Xaa⁵-Xaa⁶-Cys⁷-Xaa⁸-Xaa⁹-Xaa¹⁰-Xaa¹¹-Cys¹²-Xaa¹³-Xaa¹⁴-Cys¹⁵-Xaa_(n2)¹⁶ 292 (II-Aad) Formula 4:12,7:15Xa_(n1)-Maa⁴-Glu⁵-Xaa⁶-Maa⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Maa¹²-Thr¹³-Gly¹⁴⁻Maa¹⁵-Xaa_(n2)293 III (III-Aad) Formula 4:12,7:15Xaa_(n1)-Maa⁴-Xaa⁵-Xaa⁶-Maa⁷-Xaa⁸-Xaa⁹-Xaa¹⁰-Xaa¹¹-Maa¹²-Xaa¹³-Xaa¹⁴-Maa¹⁵-Xaa_(n2)294 IV (IV-Aad) Formula V C4:C12,C7:C15Asn¹-Asp²-Aad³-Cys⁴-Xaa⁵-Xaa⁶-Cys⁷-Xaa⁸-Asn⁹-Xaa¹⁰-Xaa¹¹-Cys¹²-Xaa¹³-Xaa¹⁴-Cys¹⁵-Xaa¹⁶295 (V-Aad) Formula C4:C12,C7:C15dAsn¹-Glu²-Aad³-Cys⁴-Xaa⁵-Xaa⁶-Cys⁷-Xaa⁸-Asn⁹-Xaa¹⁰-Xaa¹¹-Cys¹²-Xaa¹³-Xaa¹⁴-Cys¹⁵-d-Xaa¹⁶296 VI (VI-Aad) Formula C4:C12,C7:C15dAsn¹-dGlu²-Aad³-Cys⁴-Xaa⁵-Xaa⁶-Cys⁷-Xaa⁸-Asn⁹-Xaa¹⁰-Aaa¹¹-Cys¹²-Xaa¹³-Xaa¹⁴-Cys¹⁵-d-Xaa¹⁶297 VII-a (VI-a- Aad) Formula C4:C12,C7:C15dAsn¹-dAsp²-Aad³-Cys⁴-Xaa⁵-Xaa⁶-Cys⁷-Xaa⁸-Asn⁹-Xaa¹⁰-Xaa¹¹-Cys¹²-Xaa¹³-Xaa¹⁴-Cys¹⁵-d-Xaa¹⁶298 VII-b (VI-b- Aad) Formula C4:C12,C7:C15dAsn¹-dAsp²-Aad³-Cys⁴-Xaa⁵-Xaa⁶-Cys⁷-Xaa⁸-Tyr⁹-Xaa¹⁰-Xaa¹¹-Cys¹²-Xaa¹³-Xaa¹⁴-Cys¹⁵-d-Xaa¹⁶299 VIII (VIII-Aad) Formula C4:C12,C7:C15dAsn¹-dGlu²-Aad³-Cys⁴-Xaa⁵-Xaa⁶-Cys⁷-Xaa⁸-Tyr⁹-Xaa¹⁰-Xaa¹¹-Cys¹²-Xaa¹³-Xaa¹⁴-Cys¹⁵-d-Xaa¹⁶300 IX (IX-Aad) Formula C4:C11C7:C15Xaa_(n1)-Cys⁴-Xaa⁵-Xaa⁶-Xaa⁷-Xaa⁸-Xaa⁹-Xaa¹⁰-Xaa¹¹-Cys¹²-Xaa¹³-Xaa¹⁴-Xaa¹⁵-Xaa_(n2)¹⁶ 301 XXI Aad) Aad) SP-363 C4:C12, C7:C15dAsn¹-Asp²-Aad³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu-AMIDE¹⁶302 SP-364 C4:C12, C7:C15dAsn¹-Asp²-Aad³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dSer¹⁶303 SP-365 C4:C12, C7:C15dAsn¹-Asp²-Aad³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dSer-AMIDE¹⁶304 SP-366 C4:C12, C7:C15dAsn¹-Asp²-Aad³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dTyr¹⁶305 SP-367 C4:C12, C7:C15dAsn¹-Asp²-Aad³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dTyr-AMIDE¹⁶306 SP-373 C4:C11, C7:C15Pyglu¹-Asp²-Aad³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu-AMIDE¹⁶307 / C4:C12, C7:C15Pyglu¹-Asp²-Aad³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶308 SP- C4:C12, C7:C15PEG3-Asn¹-Asp²-Aad³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶-PEG3309 304diPEG SP-304N- C4:C12, C7:C15PEG3-Asn¹-Asp²-Aad³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶310 PEG SP-304C- C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Aad⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶-PEG3311 PEG Formula C4:C12, C7:C15Xaa¹-Xaa²-Aad³-Maa⁴-Xaa⁵-Xaa⁶-Maa⁷-Xaa⁸-Xaa⁹-Xaa¹⁰-Xaa¹¹-Maa¹²-Xaa¹³-Xaa¹⁴-Maa¹⁵-Xaa¹⁶312 XVIII (XVIII- Aad) N32 C4:C12, C7:C15Glu¹-Asp²-Aad³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶313 N34 C4:C12, C7:C15Glu¹-Glu²-Aad³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶314 N36 C4:C12, C7:C15Asp¹-Asp²-Aad³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶315 N38 C4:C12, C7:C15Asp¹-Glu²-Aad³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶316 N40 C4:C12, C7:C15Gln¹-Asp²-Aad³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶317 N42 C4:C12, C7:C15Gln¹-Glu²-Aad³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶318 N44 C4:C12, C7:C15Lys¹-Asp²-Aad³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶319 N46 C4:C12, C7:C15Lys¹-Glu²-Aad³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶320 N48 C4:C12, C7:C15Glu¹-Asp²-Aad³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶321 N50 C4:C12, C7:C15Glu¹-Glu²-Aad³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶322 N52 C4:C12, C7:C15Asp¹-Asp²-Aad³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶323 N54 C4:C12, C7:C15Asp¹-Glu²-Aad³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶324 N56 C4:C12, C7:C15Gln¹-Asp²-Aad³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶325 N58 C4:C12, C7:C15Gln¹-Glu²-Aad³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶326 N60 C4:C12, C7:C15Lys¹-Asp²-Aad³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶327 N62 C4:C12, C7:C15Lys¹-Glu²-Aad³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶328 N65 C4:C12, C7:C15Glu¹-Asp²-Aad³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶329 N67 C4:C12, C7:C15Glu¹-Glu²-Aad³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶330 N69 C4:C12, C7:C15Asp¹-Asp²-Aad³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶331 N71 C4:C12, C7:C15Asp¹-Glu²-Aad³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶332 N73 C4:C12, C7:C15Gln¹-Asp²-Aad³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶333 N75 C4:C12, C7:C15Gln¹-Glu²-Aad³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶334 N77 C4:C12, C7:C15Lys¹-Asp²-Aad³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶335 N79 C4:C12, C7:C15Lys¹-Glu²-Aad³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶336 N81 C4:C12, C7:C15Glu¹-Asp²-Aad³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶337 N83 C4:C12, C7:C15Glu¹-Glu²-Aad³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶338 N85 C4:C12, C7:C15Asp¹-Asp²-Aad³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶339 N87 C4:C12, C7:C15Asp¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶340 N88 C4:C12, C7:C15Asp¹-Glu²-Aad³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶341 N89 C4:C12, C7:C15Gin¹-Asp²-Aad³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶342 N91 C4:C12, C7:C15Gln¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶343 N92 C4:C12, C7:C15Gin¹-Glu²-Aad³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶344 N93 C4:C12, C7:C15Lys¹-Asp²-Aad³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶345 N95 C4:C12, C7:C15Lys¹-Glu²-Aad³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶346

The GCRA peptides and derivative or analogs thereof described hereinbind the guanylate cyclase C (GC-C) and stimulate intracellularproduction of cyclic guanosine monophosphate (cGMP). In some aspects,the GCRA peptides stimulate intracellular cGMP production at higherlevels than naturally occurring GC-C agonists (e.g., uroguanylin,guanylin, lymphoguanylin and ST peptides).

For example, the GCRA peptides of the invention stimulate 5, 10%, 20%,30%, 40%, 50%, 75%, 90% or more intracellular cGMP compared to naturallyoccurring GC-C agonists. The terms induced and stimulated are usedinterchangeably throughout the specification. The GCRA peptidesdescribed herein are more stable than naturally occurring GC-C agonists.By more stable it is meant that the peptide degrades less and/or moreslowly in simulated gastrointestinal fluid and/or simulated intestinalfluid compared to naturally occurring GC-C agonists. For example, theGCRA peptide of the invention degrade 2%, 3%, 5%, 10%, 15%, 20%, 30%,40%, 50% , 75%, 90% or less compared to naturally occurring GC-Cagonists.

As used herein, the term “guanylate cyclase receptor (GCR)” refers tothe class of guanylate cyclase C receptor on any cell type to which theinventive agonist peptides or natural agonists described herein bind. Asused herein, “intestinal guanylate cyclase receptor” is foundexclusively on epithelial cells lining the GI mucosa. Uroguanylin,guanylin, and ST peptides are expected to bind to these receptors andmay induce apoptosis. The possibility that there may be differentreceptors for each agonist peptide is not excluded. Hence, the termrefers to the class of guanylate cyclase receptors on epithelial cells.

As used herein, the term “GCC agonist” is meant to refer to peptidesand/or other compounds that bind to an intestinal guanylate cyclasereceptor and stimulate fluid and electrolyte transport. This term alsocovers fragments and pro-peptides that bind to GCR and stimulate fluidand water secretion.

As used herein, the term “substantially equivalent” is meant to refer toa peptide that has an amino acid sequence equivalent to that of thebinding domain where certain residues may be deleted or replaced withother amino acids without impairing the peptide's ability to bind to anintestinal guanylate cyclase receptor and stimulate fluid andelectrolyte transport.

Addition of carriers (e.g., phosphate-buffered saline or PBS) and othercomponents to the composition of the present invention is well withinthe level of skill in this art. In addition to the compound, suchcompositions may contain pharmaceutically acceptable carriers and otheringredients known to facilitate administration and/or enhance uptake.Other formulations, such as microspheres, nanoparticles, liposomes, andimmunologically-based systems may also be used in accordance with thepresent invention. Other examples include formulations with polymers(e.g., 20% w/v polyethylene glycol) or cellulose, or entericformulations.

GCRA Peptides

In one aspect, the invention provides a GCRA peptide. The GCRA peptidesare analogues uroguanylin, guanylin, lymphoguanylin and ST peptides. Noparticular length is implied by the term “peptide”. In some embodiments,the GCRA peptide is less than 25 amino acids in length, e.g., less thanor equal to 20, 15, 14, 13, 12, 11, 10, or 5 amino acid in length.

The GCRA peptides can be polymers of L-amino acids, D-amino acids, or acombination of both. For example, in various embodiments, the peptidesare D retro-inverso peptides. The term “retro-inverso isomer” refers toan isomer of a linear peptide in which the direction of the sequence isreversed and the chirality of each amino acid residue is inverted. See,e.g., Jameson et al., Nature, 368, 744-746 (1994); Brady et al., Nature,368, 692-693 (1994). The net result of combining D-enantiomers andreverse synthesis is that the positions of carbonyl and amino groups ineach amide bond are exchanged, while the position of the side-chaingroups at each alpha carbon is preserved. Unless specifically statedotherwise, it is presumed that any given L-amino acid sequence of theinvention may be made into a D retro-inverso peptide by synthesizing areverse of the sequence for the corresponding native L-amino acidsequence. For example a GCRA peptide includes the sequence defined byFormulae I-XXI, their corresponding α-aminoadipic acid (Aad) derivatives(e.g., Formula I-Aad, II-Aad, III-Aad, IV-Aad, V-Aad, VI-Aad, VII-a-Aad,VII-b-Aad, VIII-Aad, IX-Aad, XVIII-Aad or XXI-Aad), as well as thoseamino acid sequence summarized below in Tables 1-8.

By inducing cGMP production is meant that the GCRA peptide induces theproduction of intracellular cGMP. Intracellular cGMP is measured bymethods known in the art. For example, the GCRA peptide of the inventionstimulate 5%, 10%, 20%, 30%, 40%, 50% , 75%, 90% or more intracellularcGMP compared to naturally occurring GC-C agonists. In some embodimentsthe GCRA peptides described herein are more stable than naturallyoccurring GC-C agonists. By more stable it is meant that the peptidedegrade less and/or more slowly in simulated gastric fluid and/orsimulated intestinal fluid compared to naturally occurring GC-Cagonists. For example, the GCRA peptide of the invention degrade 2%, 3%,5%, 10%, 15%, 20%, 30%, 40%, 50% , 75%, 90% or less compared tonaturally occurring GC-C agonists.

As used herein PEG3, 3 PEG, is meant to denote polyethylene glycol suchas include aminoethyloxy-ethyloxy-acetic acid (AeeA).

As used herein, the term “AMIDE” is meant to denote that the terminalcarboxylic acid is replaced with an amide group, i.e., the terminal COOHis replaced with CONH₂.

As used herein (e.g., in Formulae I-XXI, their correspondingα-aminoadipic acid (Aad) derivatives represented by Formulae I-Aad,II-Aad, III-Aad, IV-Aad, V-Aad, VI-Aad, VII-a-Aad, VII-b-Aad, VIII-Aad,IX-Aad, XVIII-Aad or XXI-Aad), X_(aa) is any natural, unnatural aminoacid or amino acid analogue; M_(aa) is a Cysteine (Cys), Penicillamine(Pen), homocysteine, or 3-mercaptoproline. Xaa_(n1) is meant to denotean amino acid sequence of any natural, unnatural amino acid or aminoacid analogue that is one, two or three residues in length; Xaa_(n2)ismeant to denote an amino acid sequence of any natural, unnatural aminoacid or amino acid analogue that is zero or one residue in length; andXaa_(n3) is meant to denote an amino acid sequence of any natural,unnatural amino acid or amino acid analogue that is zero, one, two,three, four, five or six residues in length. Additionally, any aminoacid represented by Xaa, may be an L-amino acid, a D-amino acid, amethylated amino acid, a fluorinated amino acid or any combination ofthereof. Preferably the amino acid at the N-terminus, C-terminus or bothis a D-amino acid. Optionally, any GCRA peptide represented by FormulaeI-XXI and their corresponding α-aminoadipic acid (Aad) derivativesrepresented by Formulae I-Aad, II-Aad, III-Aad, IV-Aad, V-Aad, VI-Aad,VII-a-Aad, VII-b-Aad, VIII-Aad, IX-Aad, XVIII-Aad or XXI-Aad may containon or more polyethylene glycol residues at the N-terminus, C-terminus orboth. An exemplary polyethylene glycol includesaminoethyloxy-ethyloxy-acetic acid and polymers thereof. In someembodiments, any GCRA peptide represented by Formulae I-XXI and theircorresponding α-aminoadipic acid (Aad) derivatives represented byFormulae I-Aad, II-Aad, III-Aad, IV-Aad, V-Aad, VI-Aad, VII-a-Aad,VII-b-Aad, VIII-Aad, IX-Aad, XVIII-Aad or XXI-Aad may contain AMIDE atthe c-terminus.

Specific examples of GCRA peptides that can be used in the methods andformulations of the invention include a peptide selected from the groupdesignated by SEQ ID NOs: 1-346.

In some embodiments, GCRA peptides include peptides having the aminoacid sequence of Formula I. In some embodiments, at least one amino acidof Formula I is a D-amino acid or a methylated amino acid and/or theamino acid at position 16 is a serine. Preferably, the amino acid atposition 16 of Formula I is a D-amino acid or a methylated amino acid.For example, the amino acid at position 16 of Formula I is a d-leucineor a d-serine. Optionally, one or more of the amino acids at positions1-3 of Formula I are D-amino acids or methylated amino acids or acombination of D-amino acids or methylated amino acids. For example,Asn¹, Asp² or Glu³ (or a combination thereof) of Formula I is a D-aminoacid or a methylated amino acid. Preferably, the amino acid at positionXaa⁶ of Formula I is a leucine, serine or tyrosine.

In alternative embodiments, GCRA peptides include peptides having theamino acid sequence of Formula II. In some embodiments, at least oneamino acid of Formula II is a D-amino acid or a methylated amino acid.Preferably, the amino acid denoted by Xaa_(n2) of Formula II is aD-amino acid or a methylated amino acid. In some embodiments, the aminoacid denoted by Xaa_(n2) of Formula II is a leucine, a d-leucine, aserine, or a d-serine. Preferably, the one or more amino acids denotedby Xaa_(n1) of Formula II are D-amino acids or methylated amino acids.Preferably, the amino acid at position Xaa⁶ of Formula II is a leucine,a serine, or a tyrosine. In some embodiments, Xaa¹ is a pyroglutamicacid. In some embodiments, Xaa² is glutamic acid or d-glutamic acid. Insome embodiments, Xaa³ is an aspartic acid or d-aspartic acid. In someembodiments, Xaa⁸ and Xaa¹⁰ are AIB. In some embodiments, Xaa⁹ istyrosine. In some embodiments, Xaa¹⁶ is dNal.

In some embodiments, GCRA peptides include peptides having the aminoacid sequence of Formula III. In some embodiments, at least one aminoacid of Formula III is a D-amino acid or a methylated amino acid and/orMaa is not a cysteine. Preferably, the amino acid denoted by Xaa_(n2) ofFormula III is a D-amino acid or a methylated amino acid. In someembodiments the amino acid denoted by Xaa_(n2) of Formula III is aleucine, a d-leucine, a serine, or a d-serine. Preferably, the one ormore amino acids denoted by Xaa_(n1) of Formula III are D-amino acids ormethylated amino acids. Preferably, the amino acid at position Xaa⁶ ofFormula III is a leucine, a serine, or a tyrosine. In some embodiments,Xaa¹ is a pyroglutamic acid. In some embodiments, Xaa² is glutamic acidor d-glutamic acid. In some embodiments, Xaa³ is an aspartic acid ord-aspartic acid. In some embodiments, Xaa¹⁶ is dNal.

In other embodiments, GCRA peptides include peptides having the aminoacid sequence of Formula IV. In some embodiments, at least one aminoacid of Formula IV is a D-amino acid or a methylated amino acid, and/orMaa is not a cysteine. Preferably, the Xaa_(n2) of Formula IV is aD-amino acid or a methylated amino acid. In some embodiments, the aminoacid denoted by Xaa_(n2) of Formula IV is a leucine, a d-leucine, aserine, or a d-serine. Preferably, the one or more of the amino acidsdenoted by Xaa_(n1) of Formula IV is a D-amino acid or a methylatedamino acid. Preferably, the amino acid denoted Xaa⁶ of Formula IV is aleucine, a serine, or a tyrosine. In some embodiments, Xaa¹ is apyroglutamic acid. In some embodiments, Xaa² is glutamic acid ord-glutamic acid. In some embodiments, Xaa³ is an aspartic acid ord-aspartic acid. In some embodiments, Xaa⁸ and Xaa¹⁰ are AIB. In someembodiments, Xaa⁹ is tyrosine. In some embodiments, Xaa¹⁶ is dNal.

In further embodiments, GCRA peptides include peptides having the aminoacid sequence of Formula V. In some embodiments, at least one amino acidof Formula V is a D-amino acid or a methylated amino acid. Preferably,the amino acid at position 16 of Formula V is a D-amino acid or amethylated amino acid. For example, the amino acid at position 16 (i.e.,Xaa¹⁶) of Formula V is a d-leucine or a d-serine. Optionally, one ormore of the amino acids at position 1-3 of Formula V are D-amino acidsor methylated amino acids or a combination of D-amino acids ormethylated amino acids. For example, Asn¹, Asp² or Glu³ (or acombination thereof) of Formula V is a D-amino acids or a methylatedamino acid. Preferably, the amino acid denoted at Xaa⁶ of Formula V is aleucine, a serine, or a tyrosine.

In additional embodiments, GCRA peptides include peptides having theamino acid sequence of Formula VI, VII, VIII, or IX. Preferably, theamino acid at position 6 of Formula VI, VII, VIII, or IX is a leucine, aserine, or a tyrosine. In some aspects the amino acid at position 16 ofFormula VI, VII, VIII, or IX is a leucine or a serine. Preferably, theamino acid at position 16 of Formula V is a D-amino acid or a methylatedamino acid.

In additional embodiments, GCRA peptides include peptides having theamino acid sequence of Formula X, XI, XII, XIII, XIV, XV, XVI or XVII.Optionally, one or more amino acids of Formulae X, XI, XII, XIII, XIV,XV, XVI or XVII are D-amino acids or methylated amino acids. Preferably,the amino acid at the carboxyl terminus of the peptides according toFormulae X, XI, XII, XIII, XIV, XV, XVI or XVII is a D-amino acid or amethylated amino acid. For example the amino acid at the carboxylterminus of the peptides according to Formulae X, XI, XII, XIII, XIV,XV, XVI or XVII is a D-tyrosine.

Preferably, the amino acid denoted by Xaa⁶ of Formula XIV is a tyrosine,phenylalanine or a serine. Most preferably the amino acid denoted byXaa⁶ of Formula XIV is a phenylalanine or a serine. Preferably, theamino acid denoted by Xaa⁴ of Formula XV, XVI or XVII is a tyrosine, aphenylalanine, or a serine. Most preferably, the amino acid positionXaa⁴ of Formula V, XVI or XVII is a phenylalanine or a serine.

In some embodiments, GCRA peptides include peptides containing the aminoacid sequence of Formula XVIII. Preferably, the amino acid at position 1of Formula XVIII is a glutamic acid, aspartic acid, glutamine or lysine.Preferably, the amino acid at position 2 and 3 of Formula XVIII is aglutamic acid, or an aspartic acid. Preferably, the amino acid atposition 5 is a glutamic acid. Preferably, the amino acid at position 6of Formula XVIII is an isoleucine, valine, serine, threonine ortyrosine. Preferably, the amino acid at position 8 of Formula XVIII is avaline or isoleucine. Preferably, the amino acid at position 9 ofFormula XVIII is an asparagine. Preferably, the amino acid at position10 of Formula XVIII is a valine or a methionine. Preferably, the aminoacid at position 11 of Formula XVIII is an alanine. Preferably, theamino acid at position 13 of Formula XVIII is a threonine. Preferably,the amino acid at position 14 of Formula XVIII is a glycine. Preferably,the amino acid at position 16 of Formula XVIII is a leucine, serine orthreonine

In alternative embodiments, GCRA peptides include peptides containingthe amino acid sequence of Formula XIX. Preferably, the amino acid atposition 1 of Formula XIX is a serine or asparagine. Preferably, theamino acid at position 2 of Formula XIX is a histidine or an asparticacid. Preferably, the amino acid at position 3 of Formula XIX is athreonine or a glutamic acid. Preferably, the amino acid at position 5of Formula XIX is a glutamic acid. Preferably, the amino acid atposition 6 of Formula XIX is an isoleucine, leucine, valine or tyrosine.Preferably, the amino acid at position 8, 10, 11, or 13 of Formula XIXis an alanine. Preferably, the amino acid at position 9 of Formula XIXis an asparagine or a phenylalanine. Preferably, the amino acid atposition 14 of Formula XIX is a glycine.

In further embodiments, GCRA peptides include peptides containing theamino acid sequence of Formula XX. Preferably, the amino acid atposition 1 of Formula XX is a glutamine. Preferably, the amino acid atposition 2 or 3 of Formula XX is a glutamic acid or an aspartic acid.Preferably, the amino acid at position 5 of Formula XX is a glutamicacid. Preferably, the amino acid at position 6 of Formula XX isthreonine, glutamine, tyrosine, isoleucine, or leucine. Preferably, theamino acid at position 8 of Formula XX is isoleucine or valine.Preferably, the amino acid at position 9 of Formula XX is asparagine.Preferably, the amino acid at position 10 of Formula XX is methionine orvaline. Preferably, the amino acid at position 11 of Formula XX isalanine. Preferably, the amino acid at position 13 of Formula XX is athreonine. Preferably, the amino acid at position 1 of Formula XX is aglycine. Preferably, the amino acid at position 15 of Formula XX is atyrosine. Optionally, the amino acid at position 15 of Formula XX istwo-amino acid in length and is Cysteine (Cys), Penicillamine (Pen)homocysteine, or 3-mercaptoproline and serine, leucine or threonine.

In some embodiments, GCRA peptides include peptides having the aminoacid sequence of Formula XXI. In some embodiments, at least one aminoacid of Formula XXI is a D-amino acid or a methylated amino acid.Preferably, the amino acid denoted by Xaa_(n2) of Formula XXI is aD-amino acid or a methylated amino acid. In some embodiments, the aminoacid denoted by Xaa_(n2) of Formula XXI is a leucine, a d-leucine, aserine, or a d-serine. Preferably, the one or more amino acids denotedby Xaa_(n1) of Formula XXI are D-amino acids or methylated amino acids.Preferably, the amino acid at position Xaa⁶ of Formula XXI is a leucine,a serine, or a tyrosine. In some embodiments, Xaa¹ is a pyroglutamicacid. In some embodiments, Xaa² is glutamic acid or d-glutamic acid. Insome embodiments, Xaa³ is an aspartic acid or d-aspartic acid. In someembodiments, Xaa⁷ is an aspartic acid and forms a lactam bridge withXaa¹⁵. In some embodiments, Xaa⁸ and Xaa¹⁰ are AIB. In some embodiments,Xaa⁹ is tyrosine. In some embodiments, Xaa¹⁵ is an Orn. In someembodiments, Xaa¹⁶ is dNal.

The GCRA peptides of the invention also include analogs that contain anα-aminoadipic acid (Aad), preferably at the 3rd position from theN-terminus of each peptide or at the position to the N-terminal sidenext to the first cysteine (“Cys”) residue. In some embodiments, theGCRA peptide Aad derivatives include peptides having the amino acidsequences of Formula I-Aad, II-Aad, III-Aad, IV-Aad, V-Aad, VI-Aad,VII-a-Aad, VII-b-Aad, VIII-Aad, IX-Aad, XVIII-Aad or XXI-Aad (Table 8).Except the Aad replacement described herein, variations of amino acid ateach position of each Formula are the same as those described above inits corresponding Formula sequence without Aad. In some embodiments,when Xaa_(n1) represents one amino acid, Xaa_(n1) is an α-aminoadipicacid (Aad). In some embodiments, when Xaa_(n1) represents two aminoacids, the second residue from the N-terminus is an α-aminoadipic acid(Aad). In some embodiments, when Xaa_(n1) represents three amino acids,the third residue from the N-terminus is an α-aminoadipic acid (Aad).Exemplary Ad analogs are listed in Table 8.

In certain embodiments, one or more amino acids of the GCRA peptides canbe replaced by a non-naturally occurring amino acid or a naturally ornon-naturally occurring amino acid analog. There are many amino acidsbeyond the standard 20 (Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His,Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and VaI). Some arenaturally-occurring others are not. (See, for example, Hunt, TheNon-Protein Amino Acids: In Chemistry and Biochemistry of the AminoAcids, Barrett, Chapman and Hall, 1985). For example, an aromatic aminoacid can be replaced by 3,4-dihydroxy-L-phenylalanine,3-iodo-L-tyrosine, triiodothyronine, L-thyroxine, phenylglycine (Phg) ornor-tyrosine (norTyr). Phg and norTyr and other amino acids includingPhe and Tyr can be substituted by, e.g., a halogen, —CH3, —OH, —CH2NH3,—C(O)H, —CH2CH3, —CN, —CH2CH2CH3, —SH, or another group. Any amino acidcan be substituted by the D-form of the amino acid.

With regard to non-naturally occurring amino acids or naturally andnon-naturally occurring amino acid analogs, a number of substitutions inthe polypeptide and agonists described herein are possible alone or incombination.

For example, glutamine residues can be substituted withgamma-Hydroxy-Glu or gamma-Carboxy-Glu. Tyrosine residues can besubstituted with an alpha substituted amino acid such asL-alpha-methylphenylalanine or by analogues such as: 3-Amino-Tyr;Tyr(CH3); Tyr(PO3(CH3)2); Tyr(SO3H); beta-Cyclohexyl-Ala;beta-(1-Cyclopentenyl)-Ala; beta-Cyclopentyl-Ala; beta-Cyclopropyl-Ala;beta-Quinolyl-Ala; beta-(2-Thiazolyl)-Ala; beta-(Triazole-1-yl)-Ala;beta-(2-Pyridyl)-Ala; beta-(3-Pyridyl)-Ala; Amino-Phe; Fluoro-Phe;Cyclohexyl-Gly; tBu-Gly; beta-(3-benzothienyl)-Ala;beta-(2-thienyl)-Ala; 5-Methyl-Trp; and A-Methyl-Trp. Proline residuescan be substituted with homopro (L-pipecolic acid); hydroxy-Pro;3,4-Dehydro-Pro; 4-fluoro-Pro; or alpha-methyl-Pro or anN(alpha)-C(alpha) cyclized amino acid analogues with the structure: n=0,1, 2, 3 Alanine residues can be substituted with alpha-substituted orN-methylated amino acid such as alpha-amino isobutyric acid (aib),L/D-alpha-ethylalanine (L/D-isovaline), L/D-methylvaline, orL/D-alpha-methylleucine or a non-natural amino acid such asbeta-fluoro-Ala. Alanine can also be substituted with: n=0, 1, 2, 3Glycine residues can be substituted with alpha-amino isobutyric acid(aib) or L/D-alpha-ethylalanine (L/D-isovaline).

Further examples of unnatural amino acids include: an unnatural analogof tyrosine; an unnatural analogue of glutamine; an unnatural analogueof phenylalanine; an unnatural analogue of serine; an unnatural analogueof threonine; an alkyl, aryl, acyl, azido, cyano, halo, hydrazine,hydrazide, hydroxyl, alkenyl, alkynl, ether, thiol, sulfonyl, seleno,ester, thioacid, borate, boronate, phospho, phosphono, phosphine,heterocyclic, enone, imine, aldehyde, hydroxylamine, keto, or aminosubstituted amino acid, or any combination thereof; an amino acid with aphotoactivatable cross-linker; a spin-labeled amino acid; a fluorescentamino acid; an amino acid with a novel functional group; an amino acidthat covalently or noncovalently interacts with another molecule; ametal binding amino acid; an amino acid that is amidated at a site thatis not naturally amidated, a metal-containing amino acid; a radioactiveamino acid; a photocaged and/or photoisomerizable amino acid; a biotinor biotin-analogue containing amino acid; a glycosylated or carbohydratemodified amino acid; a keto containing amino acid; amino acidscomprising polyethylene glycol or polyether; a heavy atom substitutedamino acid (e.g., an amino acid containing deuterium, tritium, ¹³C, ¹⁵N,or ¹⁸O); a chemically cleavable or photocleavable amino acid; an aminoacid with an elongated side chain; an amino acid containing a toxicgroup; a sugar substituted amino acid, e.g., a sugar substituted serineor the like; a carbon-linked sugar-containing amino acid; a redox-activeamino acid; an α-hydroxy containing acid; an amino thio acid containingamino acid; an α, α disubstituted amino acid; a β-amino acid; a cyclicamino acid other than proline; an O-methyl-L-tyrosine; anL-3-(2-naphthyl)alanine; a 3-methyl-phenylalanine; aρ-acetyl-L-phenylalanine; an O-4-allyl-L-tyrosine; a4-propyl-L-tyrosine; a tri-O-acetyl-GlcNAc β-serine; an L-Dopa; afluorinated phenylalanine; an isopropyl-L-phenylalanine; ap-azido-L-phenylalanine; a p-acyl-L-phenylalanine; ap-benzoyl-L-phenylalanine; an L-phosphoserine; a phosphonoserine; aphosphonotyrosine; a p-iodo-phenylalanine; a 4-fluorophenylglycine; ap-bromophenylalanine; a p-amino-L-phenylalanine; anisopropyl-L-phenylalanine; L-3-(2-naphthyl)alanine;D-3-(2-naphthyl)alanine (dNal); an amino-, isopropyl-, orO-allyl-containing phenylalanine analogue; a dopa, O-methyl-L-tyrosine;a glycosylated amino acid; a p-(propargyloxy)phenylalanine;dimethyl-Lysine; hydroxy-proline; mercaptopropionic acid; methyl-lysine;3-nitro-tyrosine; norleucine; pyro-glutamic acid; Z (Carbobenzoxyl);ε-Acetyl-Lysine; β-alanine; aminobenzoyl derivative; aminobutyric acid(Abu); citrulline; aminohexanoic acid; aminoisobutyric acid (AIB);cyclohexylalanine; d-cyclohexylalanine; hydroxyproline; nitro-arginine;nitro-phenylalanine; nitro-tyrosine; norvaline; octahydroindolecarboxylate; ornithine (Orn); penicillamine (PEN);tetrahydroisoquinoline; acetamidomethyl protected amino acids andpegylated amino acids. Further examples of unnatural amino acids andamino acid analogs can be found in U.S. 20030108885, U.S. 20030082575,US20060019347 (paragraphs 410-418) and the references cited therein. Thepolypeptides of the invention can include further modificationsincluding those described in US20060019347, paragraph 589.

In some embodiments, an amino acid can be replaced by anaturally-occurring, non-essential amino acid, e.g., taurine.

Alternatively, the GCRA peptides are cyclic peptides. GCRA cyclicpeptides are prepared by methods known in the art. For example,macrocyclization is often accomplished by forming an amide bond betweenthe peptide N- and C-termini, between a side chain and the N- orC-terminus [e.g., with K₃Fe(CN)₆ at pH 8.5] (Samson et al.,Endocrinology, 137: 5182-5185 (1996)), or between two amino acid sidechains, such as cysteine. See, e.g., DeGrado, Adv Protein Chem, 39:51-124 (1988). In various aspects the GCRA peptides are [4,12; 7,15]bicycles.

In some GCRA peptides one or both members of one or both pairs of Cysresidues which normally form a disulfide bond can be replaced byhomocysteine, penicillamine, 3-mercaptoproline (Kolodziej et al. 1996Int J Pept Protein Res 48:274); β,β dimethylcysteine (Hunt et al. 1993Int J Pept Protein Res 42:249) or diaminopropionic acid (Smith et al.1978 J Med Chem 2 1:117) to form alternative internal cross-links at thepositions of the normal disulfide bonds.

In addition, one or more disulfide bonds can be replaced by alternativecovalent cross-links, e.g., an amide linkage (—CH2CH(O)NHCH 2- or—CH2NHCH(O)CH 2-), an ester linkage, a thioester linkage, a lactambridge , a carbamoyl linkage, a urea linkage, a thiourea linkage, aphosphonate ester linkage, an alkyl linkage (—CH2CH2CH2CH2-), an alkenyllinkage(—CH2CH═CHCH2-), an ether linkage (—CH2CH2OCH2- or —CH2OCH2CH2-),a thioether linkage (—CH2CH2SCH2- or —CH2SCH2CH2-), an amine linkage(—CH2CH2NHCH2- or —CH2NHCH2CH2-) or a thioamide linkage (—CH2CH(S)HNHCH2- or —CH2NHCH(S)CH2-). For example, Ledu et al. (Proc Nat'l Acad. Sci.100:11263-78, 2003) describe methods for preparing lactam and amidecross-links. Exemplary GCRA peptides which include a lactam bridgeinclude for example SP-370.

The GCRA peptides can have one or more conventional polypeptide bondsreplaced by an alternative bond. Such replacements can increase thestability of the polypeptide. For example, replacement of thepolypeptide bond between a residue amino terminal to an aromatic residue(e.g. Tyr, Phe, Trp) with an alternative bond can reduce cleavage bycarboxy peptidases and may increase half-life in the digestive tract.Bonds that can replace polypeptide bonds include: a retro-inverso bond(C(O)—NH instead of NH—C(O); a reduced amide bond (NH—CH2); athiomethylene bond (S—CH2 or CH2-S); an oxomethylene bond (O—CH 2 orCH2-O); an ethylene bond (CH2-CH2); a thioamide bond (C(S)—NH); atrans-olefine bond (CH═CH); a fluoro substituted trans-olefine bond(CF═CH); a ketomethylene bond (C(O)—CHR or CHR—C(O) wherein R is H orCH3; and a fluoro-ketomethylene bond (C(O)—CFR or CFR—C(O) wherein R isH or F or CH3.

The GCRA peptides can be modified using standard modifications.Modifications may occur at the amino (N—), carboxyl (C—) terminus,internally or a combination of any of the preceding. In one aspectdescribed herein, there may be more than one type of modification on thepolypeptide. Modifications include but are not limited to: acetylation,amidation, biotinylation, cinnamoylation, farnesylation, formylation,myristoylation, palmitoylation, phosphorylation (Ser, Tyr or Thr),stearoylation, succinylation, sulfurylation and cyclisation (viadisulfide bridges or amide cyclisation), and modification by Cys3 orCys5. The GCRA peptides described herein may also be modified by2,4-dinitrophenyl (DNP), DNP-lysine, modification by7-Amino-4-methyl-coumarin (AMC), flourescein, NBD(7-Nitrobenz-2-Oxa-1,3-Diazole), p-nitro-anilide, rhodamine B, EDANS(5-((2-aminoethyl)amino)naphthalene-1-sulfonic acid), dabcyl, dabsyl,dansyl, texas red, FMOC, and Tamra (Tetramethylrhodamine). The GCRApeptides described herein may also be conjugated to, for example,polyethylene glycol (PEG); alkyl groups (e.g., C1-C20 straight orbranched alkyl groups); fatty acid radicals; combinations of PEG, alkylgroups and fatty acid radicals (See, U.S. Pat. No. 6,309,633; Soltero etal., 2001 Innovations in Pharmaceutical Technology 106-110); BSA and KLH(Keyhole Limpet Hemocyanin). The addition of PEG and other polymerswhich can be used to modify polypeptides of the invention is describedin US2006019347 section IX.

Also included in the invention are peptides that biologically orfunctional equivalent to the peptides described herein. The term“biologically equivalent” or functional equivalent” is intended to meanthat the compositions of the present invention are capable ofdemonstrating some or all of the cGMP production modulatory effects.

GCRA peptides can also include derivatives of GCRA peptides which areintended to include hybrid and modified forms of GCRA peptides in whichcertain amino acids have been deleted or replaced and modifications suchas where one or more amino acids have been changed to a modified aminoacid or unusual amino acid and modifications such as glycosylation solong the modified form retains the biological activity of GCRA peptides.By retaining the biological activity, it is meant that cGMP and orapoptosis is induced by the GCRA peptide, although not necessarily atthe same level of potency as that of a naturally-occurring GCRA peptideidentified.

Preferred variants are those that have conservative amino acidsubstitutions made at one or more predicted non-essential amino acidresidues. A “conservative amino acid substitution” is one in which theamino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). Thus, a predicted nonessential amino acid residue in a GCRApolypeptide is replaced with another amino acid residue from the sameside chain family. Alternatively, in another embodiment, mutations canbe introduced randomly along all or part of a GCRA coding sequence, suchas by saturation mutagenesis, and the resultant mutants can be screenedto identify mutants that retain activity.

Also included within the meaning of substantially homologous is any GCRApeptide which may be isolated by virtue of cross-reactivity withantibodies to the GCRA peptide.

Preparation of GCRA Peptides

GCRA peptides are easily prepared using modern cloning techniques, ormay be synthesized by solid state methods or by site-directedmutagenesis. A GCRA peptide may include dominant negative forms of apolypeptide.

Chemical synthesis may generally be performed using standard solutionphase or solid phase peptide synthesis techniques, in which a peptidelinkage occurs through the direct condensation of the amino group of oneamino acid with the carboxyl group of the other amino acid with theelimination of a water molecule. Peptide bond synthesis by directcondensation, as formulated above, requires suppression of the reactivecharacter of the amino group of the first and of the carboxyl group ofthe second amino acid. The masking substituents must permit their readyremoval, without inducing breakdown of the labile peptide molecule.

In solution phase synthesis, a wide variety of coupling methods andprotecting groups may be used (See, Gross and Meienhofer, eds., “ThePeptides: Analysis, Synthesis, Biology,” Vol. 1-4 (Academic Press,1979); Bodansky and Bodansky, “The Practice of Peptide Synthesis,” 2ded. (Springer Verlag, 1994)). In addition, intermediate purification andlinear scale up are possible. Those of ordinary skill in the art willappreciate that solution synthesis requires consideration of main chainand side chain protecting groups and activation method. In addition,careful segment selection is necessary to minimize racemization duringsegment condensation. Solubility considerations are also a factor. Solidphase peptide synthesis uses an insoluble polymer for support duringorganic synthesis. The polymer-supported peptide chain permits the useof simple washing and filtration steps instead of laboriouspurifications at intermediate steps. Solid-phase peptide synthesis maygenerally be performed according to the method of Merrifield et al., J.Am. Chem. Soc., 1963, 85:2149, which involves assembling a linearpeptide chain on a resin support using protected amino acids. Solidphase peptide synthesis typically utilizes either the Boc or Fmocstrategy, which is well known in the art.

Those of ordinary skill in the art will recognize that, in solid phasesynthesis, deprotection and coupling reactions must go to completion andthe side-chain blocking groups must be stable throughout the synthesis.In addition, solid phase synthesis is generally most suitable whenpeptides are to be made on a small scale.

Acetylation of the N-terminal can be accomplished by reacting the finalpeptide with acetic anhydride before cleavage from the resin.C-amidation is accomplished using an appropriate resin such asmethylbenzhydrylamine resin using the Boc technology.

Alternatively the GCRA peptides are produced by modern cloningtechniques. For example, the GCRA peptides are produced either inbacteria including, without limitation, E. coli, or in other existingsystems for polypeptide or protein production (e.g., Bacillus subtilis,baculovirus expression systems using Drosophila Sf9 cells, yeast orfilamentous fungal expression systems, mammalian cell expressionsystems), or they can be chemically synthesized. If the GCRA peptide orvariant peptide is to be produced in bacteria, e.g., E. coli, thenucleic acid molecule encoding the polypeptide may also encode a leadersequence that permits the secretion of the mature polypeptide from thecell. Thus, the sequence encoding the polypeptide can include the presequence and the pro sequence of, for example, a naturally-occurringbacterial ST polypeptide. The secreted, mature polypeptide can bepurified from the culture medium.

The sequence encoding a GCRA peptide described herein can be insertedinto a vector capable of delivering and maintaining the nucleic acidmolecule in a bacterial cell. The DNA molecule may be inserted into anautonomously replicating vector (suitable vectors include, for example,pGEM3Z and pcDNA3, and derivatives thereof). The vector nucleic acid maybe a bacterial or bacteriophage DNA such as bacteriophage lambda or M13and derivatives thereof. Construction of a vector containing a nucleicacid described herein can be followed by transformation of a host cellsuch as a bacterium. Suitable bacterial hosts include but are notlimited to, E. coli, B subtilis, Pseudomonas, Salmonella. The geneticconstruct also includes, in addition to the encoding nucleic acidmolecule, elements that allow expression, such as a promoter andregulatory sequences. The expression vectors may contain transcriptionalcontrol sequences that control transcriptional initiation, such aspromoter, enhancer, operator, and repressor sequences.

A variety of transcriptional control sequences are well known to thosein the art. The expression vector can also include a translationregulatory sequence (e.g., an untranslated 5′ sequence, an untranslated3′ sequence, or an internal ribosome entry site). The vector can becapable of autonomous replication or it can integrate into host DNA toensure stability during polypeptide production.

The protein coding sequence that includes a GCRA peptide describedherein can also be fused to a nucleic acid encoding a polypeptideaffinity tag, e.g., glutathione S-transferase (GST), maltose E bindingprotein, protein A, FLAG tag, hexa-histidine, myc tag or the influenzaHA tag, in order to facilitate purification. The affinity tag orreporter fusion joins the reading frame of the polypeptide of interestto the reading frame of the gene encoding the affinity tag such that atranslational fusion is generated. Expression of the fusion gene resultsin translation of a single polypeptide that includes both thepolypeptide of interest and the affinity tag. In some instances whereaffinity tags are utilized, DNA sequence encoding a protease recognitionsite will be fused between the reading frames for the affinity tag andthe polypeptide of interest.

Genetic constructs and methods suitable for production of immature andmature forms of the GCRA peptides and variants described herein inprotein expression systems other than bacteria, and well known to thoseskilled in the art, can also be used to produce polypeptides in abiological system.

The peptides disclosed herein may be modified by attachment of a secondmolecule that confers a desired property upon the peptide, such asincreased half-life in the body, for example, pegylation. Suchmodifications also fall within the scope of the term “variant” as usedherein.

Compositions

The present invention also provides compositions comprising at least oneGCRA peptide (i.e., GCC agonist peptide) described herein, at least oneenteric coating which releases the peptide at a specific pH (e.g., aboutpH 4.0, pH 5.0, pH 6.0 or pH 7.0) and an inert carrier.

A composition may comprise an enteric coating which releases the peptideat pH 5 and an inert carrier coated with GCRA peptides.

A composition may comprise an enteric coating which releases the peptideat pH 6 and an inert carrier coated with GCRA peptides.

A composition may comprise an enteric coating which releases the peptideat pH 7 and an inert carrier coated with GCRA peptides.

The present invention further provides a formulation comprising amixture of compositions that contain different peptides and/or thatrelease the peptides at different pH levels. The mixture may comprise atleast 2, 3, 4 or more compositions that release the peptides atdifferent pH levels. The mixture may comprise at least 2, 3, 4 or morecompositions that contain different GCRA peptides. A skilled artisan candetermine the ratio of these compositions within the mixture, forexample, according to the activity of each peptide, solubility of eachpeptide, and/or the targeting region of the GI tract.

In some embodiments, a formulation comprises a mixture of (1) acomposition having an inert carrier coated with GCRA peptides and anenteric coating that releases the peptides at pH 5.0 (“pH 5.0composition”) and (2) a composition having an inert carrier coated withGCRA peptides and an enteric coating that releases the peptides at pH6.0 (“pH 6.0 composition”).

The ratio of pH 5.0 composition to pH 6.0 composition can be any valuebetween 100:1 (v/v) and 1:100 (v/v) and can be determined, for example,by the activity of each peptide, solubility of each peptide, and/or thetargeting region of the GI tract. In some embodiments, the ratio of pH5.0 composition to pH 6.0 composition is 10:1, 9:1, 8:1, 7:1, 6:1, 5:1,4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10.

In some embodiments, a formulation comprises a mixture of (1) acomposition having an inert carrier coated with GCRA peptides and anenteric coating that releases the peptides at pH 5.0 (“pH 5.0composition”) and (2) a composition having an inert carrier coated withGCRA peptides and an enteric coating that releases the peptides at pH7.0 (“pH 7.0 composition”).

The ratio of pH 5.0 composition to pH 7.0 composition can be any valuebetween 100:1 (v/v) and 1:100 (v/v) and can be determined, for example,by the activity of each peptide, solubility of each peptide, and/or thetargeting region of the GI tract. In some embodiments, the ratio of pH5.0 composition to pH 7.0 composition is 10:1, 9:1, 8:1, 7:1, 6:1, 5:1,4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10.

In some embodiments, a formulation comprises a mixture of (1) acomposition having an inert carrier coated with GCRA peptides and anenteric coating that releases the peptides at pH 6.0 (“pH 6.0composition”) and (2) a composition having an inert carrier coated withGCRA peptides and an enteric coating that releases the peptides at pH7.0 (“pH 7.0 composition”).

The ratio of pH 6.0 composition to pH 7.0 composition can be any valuebetween 100:1 (v/v) and 1:100 (v/v) and can be determined, for example,by the activity of each peptide, solubility of each peptide, and/or thetargeting region of the GI tract. In some embodiments, the ratio of pH6.0 composition to pH 7.0 composition is 10:1, 9:1, 8:1, 7:1, 6:1, 5:1,4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10.

In some embodiments, a formulation comprises a mixture of (1) acomposition having an inert carrier coated with GCRA peptides and anenteric coating that releases the peptides at pH 5.0 (“pH 5.0composition”); (2) a composition having an inert carrier coated withGCRA peptides and an enteric coating that releases the peptides at pH6.0 (“pH 6.0 composition”) and (3) a composition having an inert carriercoated with GCRA and an enteric coating that releases the peptides at pH7.0 (“pH 7.0 composition”).

The ratio of pH 5.0 composition to pH 6.0 composition to pH 7.0composition can be determined, for example, by the activity of eachpeptide, solubility of each peptide, and/or the targeting region of theGI tract.

In some embodiments, a formulation comprises a mixture of (1) acomposition having an inert carrier coated with GCRA peptides and anenteric coating that releases the peptides at duodenum or jejunum(“duodenum composition”) and (2) a composition having an inert carriercoated with GCRA peptides and an enteric coating that releases thepeptides at ileum, terminal ileum, or ascending colon (“ileumcomposition”).

In some embodiments, a formulation comprises a mixture of (1) acomposition having an inert carrier coated with GCRA peptides and anenteric coating that releases the peptides in a pH range of 4.5 to 5.5or in a pH range of 5.5 to 6.5 at duodenum or jejunum (“duodenumcomposition”); and (2) a composition having an inert carrier coated withGCRA peptides and an enteric coating that releases the peptides in a pHrange of 5.5 to 6.5 or in a pH range of 6.5 to 7.5 at ileum, terminalileum, or ascending colon (“ileum composition”).

The ratio of duodenum composition to ileum composition can be any valuebetween 100:1 (v/v) and 1:100 (v/v) and can be determined, for example,by the activity of each peptide, solubility of each peptide, and/or thetargeting region of the GI tract. In some embodiments, the ratio ofduodenum composition to ileum composition is 10:1, 9:1, 8:1, 7:1, 6:1,5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or1:10.

The targeting region of the GI tract includes, but is not limited to,duodenum, jejunum, ileum, terminal ileum, and ascending colon.

The GCRA peptides that can be used in the methods and formulations ofthe invention include a peptide selected from the group designated bySEQ ID NOs: 1-346. Preferably, the GCRA peptide is SEQ ID NO: 1, 9, 55or 56. In some embodiments, the inert carrier is selected from the groupconsisting of sorbitol, mannitol, EMDEX, and starch. In someembodiments, the carrier is mannitol (e.g., MANNOGEM) ormicrocrystalline cellulose (e.g., PROSOLV, CELPHERE®, CELPHERE® beads).In a preferred embodiment, the carrier is microcrystalline cellulosespheres or spherical microcrystalline cellulose, such as Celphere®SCP-100.

The enteric coating material is chosen to target the release of thecomposition of the present invention to a specific region of thegastrointestinal tract. The enteric coating material preferablycomprises one of the following: (1) a pH dependent polymer; (2) aswellable polymer; or (3) a degradable composition.

In some embodiments, the enteric coating material is an enteric coatingwhich releases the peptides at pH 5.

In some embodiments, the enteric coating material is an enteric coatingwhich releases the peptides at pH 6.

In some embodiments, the enteric coating material is an enteric coatingwhich releases the peptides at pH 7.

In accordance with the invention, the enteric coating chosen for theformulation is any coating which will achieve the targeting objective ofthe formulation. Examples of suitable enteric coatings include, but arenot limited to, the following: (1) acrylic polymers (anionic polymers ofmethacrylic acid and methacrylates polymers with methacrylic acid as afunctional group) such as the EUDRAGIT® (Degussa) polymers, e.g., forrelease in the duodenum (dissolution above pH 5.5), EUDRAGIT® L 100-55and EUDRAGIT® L 30 D-55; for release in the jejunum (dissolution abovepH 6.0), EUDRAGIT® L 100; for release in the ileum (dissolution above pH7), EUDRAGIT® S 100 and EUDRAGIT® FS 30, and COLORCON ACRYL-EZE®; (2)polyvinyl Acetate Phthalate (PVAP) including the COLORCON SURETERIC®Aqueous Enteric Coating System, and the COLORCON OPADRY® Enteric CoatingSystem; (3) hypromellose Phthalate, NF (Hydroxy Propyl Methyl CellulosePhthtalate; HPMCP; HP-55 Shin-Etsu); (4) cellulose acetate phthalate(CAP), such as AQUACOAT® CPD; and (5) cellulose acetate trimellitate(CAT). Further examples of suitable enteric coatings include, withoutlimitation, sustained release blends such as EUDRACOL, EUDRAPULSE, andEUDRAMODE, as well as sustained release polymers such as the EUDRAGIT®RL, RS, and NE polymers.

In certain embodiments, the formulations of the invention comprise apH-dependent targeting material that is pharmacologically inactive,meaning that it is excreted without being absorbed or metabolized. Insome embodiments, the GCRA peptide-loaded composition is coated with apH-dependent material. In some embodiments, the GCRA peptide-loadedcomposition is formed as a matrix with a pH-dependent material.Preferably, the pH-dependent material comprises a pH-dependent polymer.

Preferably, the pH-dependent polymer is stable in the low pH environmentof the stomach (i.e., at pH 1-2) and begins to disintegrate at thehigher pH of the small intestine (pH 6-7) or distal ileum (pH 7-8). Incertain embodiments, the polymer begins to disintegrate at pH 4.5-4.8,pH 4.8-5.0, pH 5.0-5.2, pH 5.2-5.4, pH 5.4-5.8, pH 5.8-6.0, pH 6.0-6.2,pH 6.2-6.4, pH 6.4-6.6, pH 6.6-6.8, pH 6.8-7.0, pH 7.0-7.2, or pH7.2-7.4. In certain embodiments, the polymer begins to disintegrate atpH 4.5-5.5, pH 5.5-6.5, or pH 6.5-7.5. The pH at which a pH-sensitivepolymer begins to disintegrate is also referred to herein as the“threshold pH” of the polymer.

In certain embodiments, the pH-dependent polymer is a methacrylic acidcopolymer, a polyvinyl acetate phthalate, a hydroxypropylmethylcellulosephthalate, a cellulose acetate trimelliate, a cellulose acetatephthalate, or a hydroxypropyl methyl cellulose acetate succinate.

In a preferred embodiment, the pH-dependent polymer is a methacrylicacid copolymer selected from among the EUDRAGIT® polymers. EUDRAGIT®polymers are available in a wide range of different concentrations andphysical forms, including aqueous solutions, aqueous dispersion, organicsolutions, and solid substances. The pharmaceutical properties of thepolymers are determined by the chemical properties of their functionalgroups. For example, EUDRAGIT® L, S, FS and E polymers have acidic oralkaline groups that are pH-dependent. Enteric EUDRAGIT® coatingsprovide protection against release of the GCRA peptide in the stomachand enable controlled release in the intestine. In certain embodiments,anionic EUDRAGIT® grades containing carboxyl groups are mixed with eachother to provide pH-dependent release of the GCRA peptide. In certainembodiments, EUDRAGIT® L and S grades are used for enteric coatings. Inone embodiment, EUDRAGIT® FS 30D is used for controlled release in thecolon. The various EUDRAGIT® polymers are further described ininternational pharmacopeias such as Ph. Eur., USP/NF, DMF and JPE.

In specific embodiments, the pH-dependent polymer is a methacrylic acidcopolymer selected from EUDRAGIT® L100, having a threshold pH of 6.0;EUDRAGIT® S100, having a threshold pH of 7.0; EUDRAGIT® L-30D, having athreshold pH of 5.6; EUDRAGIT® FS 30D, having a threshold pH of 6.8; orEUDRAGIT® L100-55, having a threshold pH of 5.5, or a combinationthereof.

In one embodiment, the GCRA peptide formulation comprises a targetingmaterial which provides a controlled (time-dependent) release of theGCRA peptide. Controlled release in this context includes delayedsustained release, delayed controlled release, delayed slow release,delayed prolonged release, delayed extended release, and a suddenrelease or “burst.”

Preferably, the controlled release formulation comprises a slowlydisintegrating composition comprising the GCRA peptide surrounded by thetargeting material. The targeting material preferably comprises at leastone swellable polymer. Non-limiting examples of swellable polymers foruse in a controlled release formulation of the invention include acryliccopolymers, e.g., EUDRAGIT® RL, EUDRAGIT® RS, or EUDRAGIT® NE;polyvinylacetate, e.g., KOLLICOAT® SR 30D; and cellulose derivativessuch as ethylcellulose or cellulose acetate, e.g., SURELEASE® andAQUACOAT® ECD. In a preferred embodiment, the targeting materialcomprises one or more of EUDRAGIT® RL, EUDRAGIT® RS, or EUDRAGIT® NE toprovide controlled time release of the GCRA peptide by pH-independentswelling. In a particular embodiment, the targeting material comprisesEUDRAGIT® RL:RS (2:8) and an outing coating comprising EUDRAGIT® FS.

Further non-limiting examples of swellable polymers that can be used inthe sustained release formulations of the invention includepoly(hydroxalkyl methacrylate) having a molecular weight of from 30,000to 5,000,000; kappa-carrageenan; polyvinylpyrrolidone having a molecularweight of from 10,000 to 360,000; anionic and cationic hydrogels;polyelectrolyte complexes; poly(vinyl alcohol) having low amounts ofacetate, cross-linked with glyoxal, formaldehyde, or glutaraldehyde andhaving a degree of polymerization from 200 to 30,000; a mixturecomprising methyl cellulose, cross-linked agar and carboxymethylcellulose; a water-insoluble, water-swellable copolymer produced byforming a dispersion of finely divided maleic anhydride with styrene,ethylene, propylene, butylene or isobutylene; water-swellable polymersof N-vinyl lactams; polysaccharide, water swellable gums, high viscosityhydroxylpropylmethyl cellulose and/or mixtures thereof. In certainembodiments, the swellable polymer is selected from the group consistingof calcium pectinate, cross-linked polysaccharide, water insolublestarch, microcrystalline cellulose, water insoluble cross-linkedpeptide, water insoluble cross-linked protein, water insolublecross-linked gelatin, water insoluble cross-linked hydrolyzed gelatin,water insoluble cross-linked collagen, modified cellulose, andcross-linked polyacrylic acid. Non-limiting examples of a cross-linkedpolysaccharide include insoluble metal salts or cross-linked derivativesof alginate, pectin, xantham gum, guar gum, tragacanth gum, and locustbean gum, carrageenan, metal salts thereof, and covalently cross-linkedderivatives thereof. Non-limiting examples of modified cellulose includecross-linked derivatives of hydroxypropylcellulose,hydroxypropylmethylcellulose, hydroxyethylcellulose, methylcellulose,carboxymethylcellulose, and metal salts of carboxymethylcellulose.

In certain embodiments, the swellable composition also comprises awicking agent such as silicon dioxide. The wicking agent may also beselected from a disintegrant such as microcrystalline cellulose toenhance the speed of water uptake. Other suitable wicking agentsinclude, but are not limited to, kaolin, titanium dioxide, fumed silicondioxide, alumina, niacinamide, sodium lauryl sulfate, low molecularweight polyvinyl pyrrolidone, m-pyrol, bentonite, magnesium aluminumsilicate, polyester, polyethylene, and mixtures thereof. In certainembodiments, the targeting material, which may comprise part of thecomposition and/or form one or more layers coating the composition,optionally further comprises at least one of a lubricant, a flowpromoting agent, a plasticizer, an anti-sticking agent, surfactant,wetting agent, suspending agent and dispersing agent.

In certain embodiments, the targeting material comprises a waterinsoluble polymer and a pore-forming agent. Non-limiting examples ofpore forming agents include saccharose, sodium chloride, potassiumchloride, polyvinylpyrrolidone, and/or polyethyleneglycol, water solubleorganic acids, sugars and sugar alcohol. In certain embodiments, thepore forming agent forms part of an outer layer or coating. In otherembodiments, the pore forming agent is distributed uniformly throughoutthe water insoluble polymer.

In one embodiment, the targeting material comprises a compressioncoating. Non-limiting examples of materials that can be used as acompression coating include a gum selected from the group consisting ofxanthan gum, locust bean gum, galactans, mannans, alginates, gum karaya,pectin, agar, tragacanth, accacia, carrageenan, tragacanth, chitosan,agar, alginic acid, hydrocolloids acacia catechu, salai guggal, indianbodellum, copaiba gum, asafetida, cambi gum, Enterolobium cyclocarpum,mastic gum, benzoin gum, sandarac, gambier gum, butea frondosa (Flame ofForest Gum), myrrh, konjak mannan, guar gum, welan gum, gellan gum, taragum, locust bean gum, carageenan gum, glucomannan, galactan gum, sodiumalginate, tragacanth, chitosan, xanthan gum, deacetylated xanthan gum,pectin, sodium polypectate, gluten, karaya gum, tamarind gum, ghattigum, Accaroid/Yacca/Red gum, dammar gum, juniper gum, ester gum,ipil-ipil seed gum, gum talha (acacia seyal), and cultured plant cellgums including those of the plants of the genera: acacia, actinidia,aptenia, carbobrotus, chickorium, cucumis, glycine, hibiscus, hordeum,letuca, lycopersicon, malus, medicago, mesembryanthemum, oryza, panicum,phalaris, phleum, poliathus, polycarbophil, sida, solanum, trifolium,trigonella, Afzelia africana seed gum, Treculia africana gum, detariumgum, cassia gum, carob gum, Prosopis africana gum, Colocassia esulentagum, Hakea gibbosa gum, khaya gum, scleroglucan, and zea, as well asmixtures of any of the foregoing.

In some embodiments, the targeting material further comprises aplasticizer, a stiffening agent, a wetting agent, a suspending agent, ora dispersing agent, or a combination thereof. Non-limiting examples of aplasticizer include dibutyl sebacate, polyethylene glycol andpolypropylene glycol, dibutyl phthalate, diethyl phthalate, triethylcitrate, tributyl citrate, acetylated monoglyceride, acetyl tributylcitrate, triacetin, dimethyl phthalate, benzyl benzoate, butyl and/orglycol esters of fatty acids, refined mineral oils, oleic acid, castoroil, corn oil, camphor, glycerol and sorbitol or a combination thereof.In one embodiment, the stiffening agent comprises cetyl alcohol.Non-limiting examples of wetting agents include a poloxamer,polyoxyethylene ethers, polyoxyethylene sorbitan fatty acid esters,polyoxymethylene stearate, sodium lauryl sulfate, sorbitan fatty acidesters, benzalkonium chloride, polyethoxylated castor oil, and docusatesodium. Non-limiting examples of suspending agents include alginic acid,bentonite, carbomer, carboxymethylcellulose, carboxymethylcellulosecalcium, hydroxyethylcellulose, hydroxypropylcellulose, microcrystallinecellulose, colloidal silicon dioxide, dextrin, gelatin, guar gum,xanthan gum, kaolin, magnesium aluminum silicate, maltitol, medium chaintriglycerides, methylcellulose, polyoxyethylene sorbitan fatty acidesters, polyvinylpyrrolidinone, propylene glycol alginate, sodiumalginate, sorbitan fatty acid esters, and tragacanth. Non-limitingexamples of dispersing agents include poloxamer, polyoxyethylenesorbitan fatty acid esters and sorbitan fatty acid esters.

In certain embodiments, the targeted release formulation furthercomprises an outer enteric coating over the targeted release material.Preferably, the outer enteric coating is selected from the groupconsisting of cellulose acetate phthalate, hydroxy propyl methylcellulose acetate succinate, EUDRAGIT® L100 and EUDRAGIT® L30D-55.

In one embodiment, the GCRA peptide formulation is a time-delayedformulation designed to release the GCRA peptide in a fast burst in thecolon or small intestine (“burst formulation”). The formulationcomprises a core and an outer layer. The composition comprises at leastone GCRA peptide and at least one burst controlling agent. In certainembodiments, the composition further comprises at least one disintegrantselected from the group consisting of croscarmellose sodium,crospovidone (cross-linked PVP), sodium carboxymethyl starch (sodiumstarch glycolate), cross-linked sodium carboxymethyl cellulose(Croscarmellose), pregelatinized starch (starch 1500), microcrystallinestarch, water insoluble starch, calcium carboxymethyl cellulose, andmagnesium aluminum silicate, or a combination thereof. In otherembodiments, the composition further comprises at least one of anabsorption enhancer, a binder, a hardness enhancing agent, a bufferingagent, a filler, a flow regulating agent, a lubricant, a synergisticagent, a chelator, an antioxidant, a stabilizer and a preservative.Optionally, the composition also comprises one or more other excipients.

The burst controlling agent in the composition preferably comprises awater insoluble polymer for controlling the rate of penetration of waterinto the composition and raising the internal pressure (osmoticpressure) inside the composition. Such a burst controlling agent ispreferably able to swell upon contact with liquid. Non-limiting examplesof suitable water insoluble polymers include cross-linkedpolysaccharide, water insoluble starch, microcrystalline cellulose,water insoluble cross-linked peptide, water insoluble cross-linkedprotein, water insoluble cross-linked gelatin, water insolublecross-linked hydrolyzed gelatin, water insoluble cross-linked collagenmodified cellulose, and cross-linked polyacrylic acid. In oneembodiment, the water insoluble polymer is a cross-linked polysaccharideselected from the group consisting of insoluble metal salts orcross-linked derivatives of alginate, pectin, xanthan gum, guar gum,tragacanth gum, and locust bean gum, carrageenan, metal salts thereof,and covalently cross-linked derivatives thereof. In one embodiment, thewater insoluble polymer is modified cellulose selected from the groupconsisting of cross-linked derivatives of hydroxypropylcellulose,hydroxypropylmethylcellulose, hydroxyethylcellulose, methylcellulose,carboxymethylcellulose, and metal salts of carboxymethylcellulose. Inanother embodiment, the water insoluble polymer is selected from calciumpectinate, microcrystalline cellulose, or a combination thereof.

The outer layer comprises a water insoluble hydrophobic carrier and apore forming agent comprised of a water insoluble hydrophilic particularmatter. The pore forming agent is a water permeable agent which allowsentry of liquid into the core. Optionally, the outer layer furthercomprises at least one of a wetting agent, a suspending agent, adispersing agent, a stiffening age and a plasticizer.

In certain embodiments, the water insoluble hydrophobic carrier isselected from the group consisting of adimethylaminoethylacrylate/ethylmethacrylate copolymer, the copolymerbeing based on acrylic and methacrylic acid esters with a low content ofquaternary ammonium groups, wherein the molar ratio of the ammoniumgroups to the remaining neutral (meth)acrylic acid esters isapproximately 1:20, the polymer corresponding to USP/NF “AmmonioMethacrylate Copolymer Type A”, anethylmethacrylate/chlorotrimethylammoniumethyl methacrylate copolymer,the copolymer based on acrylic and methacrylic acid esters with a lowcontent of quaternary ammonium groups wherein the molar ratio of theammonium groups to the remaining neutral (meth)acrylic acid esters is1:40, the polymer corresponding to USP/NF “Ammonio MethacrylateCopolymer Type B”, a dimethylaminoethylmethacrylate/methylmethacrylateand butylmethacrylate copolymer, a copolymer based on neutralmethacrylic acid esters and dimethylaminoethyl methacrylate esterswherein the polymer is cationic in the presence of acids, anethylacrylate and methylacrylate/ethylmethacrylate and methylmethylacrylate copolymer, the copolymer being a neutral copolymer basedon neutral methacrylic acid and acrylic acid esters, ethylcellulose,shellac, zein, and waxes.

In certain embodiments, the water insoluble particulate matter is ahydrophilic yet water insoluble polymer, preferably selected from thegroup consisting of a water insoluble cross-linked polysaccharide, awater insoluble cross-linked protein, a water insoluble cross-linkedpeptide, water insoluble cross-linked gelatin, water insolublecross-linked hydrolyzed gelatin, water insoluble cross-linked collagen,water insoluble cross linked polyacrylic acid, water insolublecross-linked cellulose derivatives, water insoluble cross-linkedpolyvinyl pyrrolidone, micro crystalline cellulose, insoluble starch,micro crystalline starch and a combination thereof. Most preferably, thewater insoluble particulate matter is microcrystalline cellulose.

In another embodiment, the GCRA peptide containing composition comprisesa GCRA peptide covalently conjugated to a carrier molecule such that thecovalent bond between the GCRA peptide and the carrier is stable in thestomach and small intestine but labile in the lower gastrointestinaltract, especially the colon. The GCRA peptide covalently linked to acarrier molecule is referred to as the “GCC prodrug.” In certainembodiments, the GCC prodrug comprises a GCRA peptide covalentlyconjugated to a carrier molecule via an azo bond or a glycosidic bond.In other embodiments, the GCC prodrug comprises a glucuronide, acyclodextrin, a dextran ester, or a polar amino acid. In certainembodiments, the GCC prodrug is a polymeric prodrug. In one embodiment,the polymeric prodrug comprises polyamides containing azo groups.

Formulations

The formulations of the invention contain one or more GCRA peptidesdescribed herein, in combination with one or more pharmaceuticallyacceptable carriers (also referred to as diluents) and/or excipients. Ina preferred embodiment, the formulations of the invention include aninert carrier. The inert carrier is preferably non-hygroscopic. In oneembodiment, the carrier in the formulation contains few or no reducingsugars and is substantially free of contaminants including, but notlimited to, iron, peroxide, and formaldehyde. In one embodiment, thecarrier is selected from the group consisting of sorbitol, mannitol,EMDEX, and starch. In one embodiment, the carrier is mannitol (e.g.,MANNOGEM) or microcrystalline cellulose (e.g. PROSOLV, CELPHERE®,CELPHERE® beads).

The formulations of the invention comprise a mixture of the compositionsdescribed herein and one or more pharmaceutically acceptable carriers(also referred to as diluents) and/or excipients.

The remainder of the formulation is comprised of the carrier and one ormore optional excipients. In one embodiment, the amount of carrier is atleast 90% of the total weight of the formulation. In another embodiment,the amount of carrier is at least 95% or at least 98% of the totalweight of the formulation. In one embodiment, the amount of carrier isbetween 90 and 99.9% of the total weight of the formulation. In oneembodiment, the one or more optional excipients comprise a disintegrantwhich is present at 1 to 5% of the total weight of the formulation. Inone embodiment, the one or more optional excipients comprise a lubricantwhich is present at 0.02 to 5% of the total weight of the formulation.In one embodiment, the one or more optional excipients comprise an aminoacid such as leucine, isoleucine, valine, histidine, phenylalanine,alanine, glutamic acid, aspartic acid, glutamine, methionine,asparagine, tyrosine, threonine, tryptophan, or glycine, which ispresent at 0.1 to 4% (e.g., 0.1-1%) of the total weight of theformulation. In one embodiment, the molar ratio between the amino acidand the GCRA peptide is from about 2:1 to about 20:1 (e.g., 5:1). In oneembodiment, the one or more optional excipients comprise a stabilizersuch as a divalent cation salt, more specifically, a water-solubledivalent cation salt (e.g., calcium chloride, magnesium chloride, zincchloride, manganese chloride), which is present at 0.1 to 12% (e.g.,0.1-4%) of the total weight of the formulation. In one embodiment, themolar ratio between the salt and the GCRA peptide is from about 5:1 toabout 20:1 (e.g., 10:1).

The formulations may contain other additives as needed, including forexample lactose, glucose, fructose, galactose, trehalose, sucrose,maltose, raffnose, maltitol, melezitose, stachyose, lactitol,palatinite, starch, xylitol, mannitol, myoinositol, and the like, andhydrates thereof, and amino acids, for example alanine, glycine andbetaine, and polypeptides and proteins, for example albumen.

Further examples of pharmaceutically acceptable carriers and excipientsinclude, but are not limited to binders, fillers, disintegrants,lubricants, anti-microbial agents, and coating agents such as: BINDERS:corn starch, potato starch, other starches, gelatin, natural andsynthetic gums such as acacia, xanthan, sodium alginate, alginic acid,other alginates, powdered tragacanth, guar gum, cellulose and itsderivatives (e.g., ethyl cellulose, cellulose acetate, carboxymethylcellulose calcium, sodium carboxymethyl cellulose), polyvinylpyrrolidone (e.g., povidone, crospovidone, copovidone, etc), methylcellulose, Methocel, pre-gelatinized starch (e.g., STARCH 1500® andSTARCH 1500 LM®, sold by Colorcon, Ltd.), hydroxypropyl methylcellulose, microcrystalline cellulose (FMC Corporation, Marcus Hook,Pa., USA), or mixtures thereof, FILLERS: talc, calcium carbonate (e.g.,granules or powder), dibasic calcium phosphate, tribasic calciumphosphate, calcium sulfate (e.g., granules or powder), microcrystallinecellulose, powdered cellulose, dextrates, kaolin, mannitol, silicicacid, sorbitol, starch, pre-gelatinized starch, dextrose, fructose,honey, lactose anhydrate, lactose monohydrate, lactose and aspartame,lactose and cellulose, lactose and microcrystalline cellulose,maltodextrin, maltose, mannitol, microcrystalline cellulose & amp; guargum, molasses, sucrose,or mixtures thereof, DISINTEGRANTS: agar-agar,alginic acid, calcium carbonate, microcrystalline cellulose,croscarmellose sodium, crospovidone, polacrilin potassium, sodium starchglycolate, potato or tapioca starch, other starches, pre-gelatinizedstarch, clays, other algins, other celluloses, gums (like gellan),low-substituted hydroxypropyl cellulose, or mixtures thereof,LUBRICANTS: calcium stearate, magnesium stearate, mineral oil, lightmineral oil, glycerin, sorbitol, mannitol, polyethylene glycol, otherglycols, stearic acid, sodium lauryl sulfate, sodium stearyl fumarate,vegetable based fatty acids lubricant, talc, hydrogenated vegetable oil(e.g., peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil,corn oil and soybean oil), zinc stearate, ethyl oleate, ethyl laurate,agar, syloid silica gel (AEROSIL 200, W.R. Grace Co., Baltimore, Md.USA), a coagulated aerosol of synthetic silica (Deaussa Co., Piano, Tex.USA), a pyrogenic silicon dioxide (CAB-O-SIL, Cabot Co., Boston, Mass.USA), or mixtures thereof, ANTI-CAKING AGENTS: calcium silicate,magnesium silicate, silicon dioxide, colloidal silicon dioxide, talc, ormixtures thereof, ANTIMICROBIAL AGENTS: benzalkonium chloride,benzethonium chloride, benzoic acid, benzyl alcohol, butyl paraben,cetylpyridinium chloride, cresol, chlorobutanol, dehydroacetic acid,ethylparaben, methylparaben, phenol, phenylethyl alcohol,phenoxyethanol, phenylmercuric acetate, phenylmercuric nitrate,potassium sorbate, propylparaben, sodium benzoate, sodiumdehydroacetate, sodium propionate, sorbic acid, thimersol, thymo, ormixtures thereof, and COATING AGENTS: sodium carboxymethyl cellulose,cellulose acetate phthalate, ethylcellulose, gelatin, pharmaceuticalglaze, hydroxypropyl cellulose, hydroxypropyl methylcellulose(hypromellose), hydroxypropyl methyl cellulose phthalate,methylcellulose, polyethylene glycol, polyvinyl acetate phthalate,shellac, sucrose, titanium dioxide, carnauba wax, microcrystalline wax,gellan gum, maltodextrin, methacrylates, microcrystalline cellulose andcarrageenan or mixtures thereof.

The formulation can also include other excipients and categories thereofincluding but not limited to Pluronic®, Poloxamers (such as Lutrol® andPoloxamer 188), ascorbic acid, glutathione, protease inhibitors (e.g.soybean trypsin inhibitor, organic acids), pH lowering agents, creamsand lotions (like maltodextrin and carrageenans); materials for chewabletablets (like dextrose, fructose, lactose monohydrate, lactose andaspartame, lactose and cellulose, maltodextrin, maltose, mannitol,microcrystalline cellulose and guar gum, sorbitol crystalline);parenterals (like mannitol and povidone); plasticizers (like dibutylsebacate, plasticizers for coatings, polyvinylacetate phthalate); powderlubricants (like glyceryl behenate); soft gelatin capsules (likesorbitol special solution); spheres for coating (like sugar spheres);spheronization agents (like glyceryl behenate and microcrystallinecellulose); suspending/gelling agents (like carrageenan, gellan gum,mannitol, microcrystalline cellulose, povidone, sodium starch glycolate,xanthan gum); sweeteners (like aspartame, aspartame and lactose,dextrose, fructose, honey, maltodextrin, maltose, mannitol, molasses,sorbitol crystalline, sorbitol special solution, sucrose); wetgranulation agents (like calcium carbonate, lactose anhydrous, lactosemonohydrate, maltodextrin, mannitol, microcrystalline cellulose,povidone, starch), caramel, carboxymethylcellulose sodium, cherry creamflavor and cherry flavor, citric acid anhydrous, citric acid,confectioner's sugar, D&C Red No. 33, D&C Yellow #10 Aluminum Lake,disodium edetate, ethyl alcohol 15%, FD&C Yellow No. 6 aluminum lake,FD&C Blue #1 Aluminum Lake, FD&C Blue No. 1, FD&C blue no. 2 aluminumlake, FD&C Green No. 3, FD&C Red No. 40, FD&C Yellow No. 6 AluminumLake, FD&C Yellow No. 6, FD&C Yellow No. 10, glycerol palmitostearate,glyceryl monostearate, indigo carmine, lecithin, manitol, methyl andpropyl parabens, mono ammonium glycyrrhizinate, natural and artificialorange flavor, pharmaceutical glaze, poloxamer 188, Polydextrose,polysorbate 20, polysorbate 80, polyvidone, pregelatinized corn starch,pregelatinized starch, red iron oxide, saccharin sodium, sodiumcarboxymethyl ether, sodium chloride, sodium citrate, sodium phosphate,strawberry flavor, synthetic black iron oxide, synthetic red iron oxide,titanium dioxide, and white wax.

Solid oral dosage forms may optionally be treated with coating systems(e.g. Opadry® fx film coating system, for example Opadry® blue(OY-LS-20921), Opadry® white (YS-2-7063), Opadry® white (YS- 1-7040),and black ink (S-1-8 106).

The agents either in their free form or as a salt can be combined with apolymer such as polylactic-glycoloic acid (PLGA),poly-(I)-lactic-glycolic-tartaric acid (P(I)LGT) (WO 01/12233),polyglycolic acid (U.S. Pat. No. 3,773,919), polylactic acid (U.S. Pat.No. 4,767,628), poly(ε-caprolactone) and poly(alkylene oxide) (U.S.20030068384) to create a sustained release formulation. Other sustainedrelease formulations and polymers for use in the compositions andmethods of the invention are described in EP 0 467 389 A2, WO 93/24150,U.S. Pat. No. 5,612,052, WO 97/40085, WO 03/075887, WO 01/01964A2, U.S.Pat. No. 5,922,356, WO 94/155587, WO 02/074247A2, WO 98/25642, U.S. Pat.Nos. 5,968,895, 6,180,608, U.S. 20030171296, U.S. 20020176841, U.S. Pat.Nos. 5,672,659, 5,893,985, 5,134,122, 5,192,741, 5,192,741, 4,668,506,4,713,244, 5,445,832 4,931,279, 5,980,945, WO 02/058672, WO 97/26015, WO97/04744, and US20020019446. In such sustained release formulationsmicroparticles (Delie and Blanco-Prieto 2005 Molecule 10:65-80) ofpolypeptide are combined with microparticles of polymer. U.S. Pat. No.6,011,01 and WO 94/06452 describe a sustained release formulationproviding either polyethylene glycols (i.e. PEG 300 and PEG 400) ortriacetin. WO 03/053401 describes a formulation which may both enhancebioavailability and provide controlled release of the agent within theGI tract. Additional controlled release formulations are described in WO02/38129, EP 326151, U.S. Pat. No. 5,236,704, WO 02/30398, WO 98/13029;U.S. 20030064105, U.S. 20030138488A1, U.S. 20030216307A1, U.S. Pat. No.6,667,060, WO 01/49249, WO 01/49311, WO 01/49249, WO 01/49311, and U.S.Pat. No. 5,877,224 materials which may include those described inWO04041195 (including the seal and enteric coating described therein)and pH-sensitive coatings that achieve delivery in the colon includingthose described in U.S. Pat. No. 4,910,021 and WO 9001329. U.S. Pat. No.4,910,021 describes using a pH-sensitive material to coat a capsule.WO9001329 describes using pH-sensitive coatings on beads containingacid, where the acid in the bead core prolongs dissolution of thepH-sensitive coating. U.S. Pat. No. 5,175,003 discloses a dual mechanismpolymer mixture composed of pH-sensitive enteric materials andfilm-forming plasticizers capable of conferring permeability to theenteric material, for use in drug-delivery systems; a matrix pelletcomposed of a dual mechanism polymer mixture permeated with a drug andsometimes covering a pharmaceutically neutral nucleus; a membrane-coatedpellet comprising a matrix pellet coated with a dual mechanism polymermixture envelope of the same or different composition; and apharmaceutical dosage form containing matrix pellets. The matrix pelletreleases acid-soluble drugs by diffusion in acid pH and bydisintegration at pH levels of nominally about 5.0 or higher.

The GCC peptides described herein may be formulated in the pH triggeredtargeted control release systems described in WO04052339. The agentsdescribed herein may be formulated according to the methodologydescribed in any of WO03105812 (extruded hyrdratable polymers);WO0243767 (enzyme cleavable membrane translocators); WO03007913 andWO03086297 (mucoadhesive systems); WO02072075 (bilayer laminatedformulation comprising pH lowering agent and absorption enhancer);WO04064769 (amidated polypeptides); WO05063156 (solid lipid suspensionwith pseudotropic and/or thixotropic properties upon melting);WO03035029 and WO03035041 (erodible, gastric retentive dosage forms);U.S. Pat. No. 5,007,790 and U.S. Pat. No. 5,972,389 (sustained releasedosage forms); WO041 1271 1 (oral extended release compositions);WO05027878, WO02072033, and WO02072034 (delayed release compositionswith natural or synthetic gum); WO05030182 (controlled releaseformulations with an ascending rate of release); WO05048998(microencapsulation system); U.S. Pat. No. 5,952,314 (biopolymer); U.S.Pat. No. 5,108,758 (glassy amylose matrix delivery); U.S. Pat. No.5,840,860 (modified starch based delivery). JP10324642 (delivery systemcomprising chitosan and gastric resistant material such as wheat gliadinor zein); U.S. Pat. Nos. 5,866,619 and 6,368,629 (saccharide containingpolymer); U.S. Pat. No. 6,531,152 (describes a drug delivery systemcontaining a water soluble core (Ca pectinate or other water-insolublepolymers) and outer coat which bursts (e.g. hydrophobicpolymer-Eudragrit)); U.S. Pat. Nos. 6,234,464; 6,403,130 (coating withpolymer containing casein and high methoxy pectin; WO0174 175 (Maillardreaction product); WO05063206 (solubility increasing formulation); WO04019872 (transferring fusion proteins).

The GCC peptides described herein may be formulated usinggastrointestinal retention system technology (GIRES; MerrionPharmaceuticals). GIRES comprises a controlled-release dosage forminside an inflatable pouch, which is placed in a drug capsule for oraladministration. Upon dissolution of the capsule, a gas-generating systeminflates the pouch in the stomach where it is retained for 16-24 hours,all the time releasing agents described herein.

The GCC peptides described herein can also be formulated using the multimatrix system technology (MMX).

The GCC peptides described herein can be formulated in an osmotic deviceincluding the ones disclosed in U.S. Pat. Nos. 4,503,030, 5,609,590 and5,358,502. U.S. Pat. No. 4,503,030 describing an osmotic device fordispensing a drug to certain pH regions of the gastrointestinal tract.More particularly, the invention relates to an osmotic device comprisinga wall formed of a semi-permeable pH sensitive composition thatsurrounds a compartment containing a drug, with a passageway through thewall connecting the exterior of the device with the compartment. Thedevice delivers the drug at a controlled rate in the region of thegastrointestinal tract having a pH of less than 3.5, and the deviceself-destructs and releases all its drug in the region of thegastrointestinal tract having a pH greater than 3.5, thereby providingtotal availability for drug absorption. U.S. Pat. Nos. 5,609,590 and5,358,502 disclose an osmotic bursting device for dispensing abeneficial agent to an aqueous environment. The device comprises abeneficial agent and osmagent surrounded at least in part by asemi-permeable membrane. The beneficial agent may also function as theosmagent. The semi-permeable membrane is permeable to water andsubstantially impermeable to the beneficial agent and osmagent. Atrigger means is attached to the semi-permeable membrane (e.g., joinstwo capsule halves). The trigger means is activated by a pH of from 3 to9 and triggers the eventual, but sudden, delivery of the beneficialagent. These devices enable the pH-triggered release of the beneficialagent core as a bolus by osmotic bursting.

The composition of the present invention can be formulated in the formof a tablet, a capsule, granules, pellets, or crystals. In certainembodiments, the composition comprises microparticles or microspheres.In one embodiment, the composition comprises a cellulose acetatebutyrate microsphere. In some embodiments, the composition comprises oneor more layers of targeting materials. In other embodiments, thecomposition is formulated in a matrix with a targeting material. Incertain embodiments, the matrix is coated with at least one additionaltargeting material.

The GCC-agonist containing composition of the present formulations isformed according to art-recognized methods. In one embodiment, thecomposition is formed with a pellet-forming agent such asmicrocrystalline cellulose, low-substituted hydroxypropylcellulose,chitin, chitosan, or any combination or mixture thereof. Generally, anamount of pellet-forming agent that is less than 20% by weight resultsin poor sphericity and broad particle size distribution. Accordingly,the pellet-forming agent of the present formulations is preferably atleast 20% by weight. In certain embodiments, the pellet-forming agent ispresent at 20% to 95% or 50% to 90% by weight.

The formulation may further comprise one or more pharmaceuticallyacceptable excipients. Preferably, the excipients are present in anamount of 2 to 70% or 5 to 50% by weight. The term excipient broadlyrefers to a biologically inactive substance used in combination with theactive agents of the formulation. An excipient can be used, for example,as a solubilizing agent, a stabilizing agent, a diluent, an inertcarrier, a preservative, a binder, a disintegrant, a coating agent, aflavoring agent, or a coloring agent. Preferably, at least one excipientis chosen to provide one or more beneficial physical properties to theformulation, such as increased stability and/or solubility of the activeagent(s).

A “pharmaceutically acceptable” excipient is one that has been approvedby a state or federal regulatory agency for use in animals, andpreferably for use in humans, or is listed in the U.S. Pharmacopia, theEuropean Pharmacopia or another generally recognized pharmacopia for usein animals, and preferably for use in humans. Examples of excipientsinclude certain inert proteins such as albumins; hydrophilic polymerssuch as polyvinylpyrrolidone; amino acids such as aspartic acid (whichmay alternatively be referred to as aspartate), glutamic acid (which mayalternatively be referred to as glutamate), lysine, arginine, glycine,and histidine; fatty acids and phospholipids such as alkyl sulfonatesand caprylate; surfactants such as sodium dodecyl sulphate andpolysorbate; nonionic surfactants such as such as TWEEN®, PLURONICS®, orpolyethylene glycol (PEG); carbohydrates such as glucose, sucrose,mannose, maltose, trehalose, and dextrins, including cyclodextrins;polyols such as sorbitol; chelating agents such as EDTA; andsalt-forming counter-ions such as sodium. Particularly preferred arehydrophilic excipients which reduce the protein binding activity andaggregation of GCRA peptides.

In some embodiments, the GCRA peptide formulation further comprises oneor more excipients selected from among an absorption enhancer, a binder,a disintegrant, and a hardness enhancing agent. In other embodiments,the formulation further comprises one or more excipients selected fromamong a wicking agent, a stabilizer, a flow regulating agent, alubricant, an antioxidant, a chelating agent, or a sequestrate.

Non-limiting examples of suitable binders include starch,polyvinylpyrrolidone (POVIDONE), low molecular weighthydroxypropylcellulose, low molecular weighthydroxypropylmethylcellulose, low molecular weightcarboxymethylcellulose, ethylcellulose, gelatin, polyethylene oxide,acacia, dextrin, magnesium aluminum silicate, and polymethacrylates.Non-limiting examples of a disintegrant include croscarmellose sodiumcrospovidone (cross-linked PVP), sodium carboxymethyl starch (sodiumstarch glycolate), pregelatinized starch (starch 1500), microcrystallinestarch, water insoluble starch, calcium carboxymethyl cellulose, andmagnesium aluminum silicate (Veegum). In certain embodiments, a binderis selected from polyvinylpyrrolidone and sodium carboxymethylcellulose.

Non-limiting examples of a wicking agent include colloidal silicondioxide, kaolin, titanium oxide, fumed silicon dioxide, alumina,niacinamide, sodium lauryl sulfate, low molecular weight polyvinylpyrrolidone, m-pyrol, bentonite, magnesium aluminum silicate, polyester,polyethylene, and mixtures thereof. In certain embodiments, a wickingagent is selected from sodium lauryl sulfate, colloidal silicon dioxide,and low molecular weight polyvinyl pyrrolidone.

Non-limiting examples of a stabilizer include butyl hydroxyanisole,ascorbic acid, citric acid, and mixtures thereof. Preferably, thestabilizer is a basic substance which can elevate the pH of an aqueoussolution or dispersion of the formulation to at least about pH 6.8.Examples of such basic substances include, for example, antacids such asmagnesium aluminometasilicate, magnesium aluminosilicate, magnesiumaluminate, dried aluminum hydroxide, synthetic hydrotalcite, syntheticaluminum silicate, magnesium carbonate, precipitated calcium carbonate,magnesium oxide, aluminum hydroxide, and sodium hydrogencarbonate. Otherexamples include pH-regulating agents such as L-arginine, sodiumphosphate, disodium hydrogen phosphate, sodium dihydrogenphosphate,potassium phosphate, dipotassium hydrogenphosphate, potassiumdihydrogenphosphate, disodium citrate, sodium succinate, ammoniumchloride, and sodium benzoate. In certain embodiments, a stabilizer isselected from ascorbic acid and magnesium aluminometasilicate.

In an embodiment where the stabilizer is a basic substance, the basicsubstance can be an inorganic water-soluble compound or an inorganicwater-insoluble compound. Non-limiting examples of an inorganicwater-soluble compounds for use as a stabilizer include carbonate saltssuch as sodium carbonate, potassium carbonate, sodium bicarbonate, orpotassium hydrogen carbonate; phosphate salts such as anhydrous sodiumphosphate, potassium phosphate, calcium dibasic phosphate, or trisodiumphosphate; and alkali metal hydroxides, such as sodium, potassium, orlithium hydroxide. Non-limiting examples of inorganic water-insolublecompounds for use as a stabilizer include suitable alkaline compoundscapable of imparting the requisite basicity, such as those commonlyemployed in antiacid compositions, for example, magnesium oxide,magnesium hydroxide, magnesium carbonate, magnesium hydrogen carbonate,aluminum hydroxide, calcium hydroxide, or calcium carbonate; compositealuminum-magnesium compounds, such as magnesium aluminum hydroxide;silicate compounds such as magnesium aluminum silicate (Veegum F),magnesium aluminometasilicate (Nesulin FH2), magnesium aluminosilicate(Nisulin A); and pharmaceutically acceptable salts of phosphoric acidsuch as tribasic calcium phosphate.

Non-limiting examples of a flow regulating agents include a colloidalsilicon dioxide and aluminum silicate.

Non-limiting examples of a lubricant include stearate salts, such asmagnesium stearate, calcium stearate, and sodium stearate, stearic acid,talc, sodium stearyl fumarate, sodium lauryl sulfate, sodium benzoate,polyethylene glycol, polyvinyl alcohol, glycerol behenate compritol(glycerol behenate), corola oil, glyceryl palmitostearate, hydrogenatedvegetable oil, magnesium oxide, mineral oil, poloxamer, and combinationsthereof. In certain embodiments, a lubricant is selected from talc andmagnesium stearate.

Non-limiting examples of antioxidants include 4,4 (2,3 dimethyltetramethylene dipyrochatechol), tocopherol-rich extract (naturalvitamin E), α-tocopherol, β-tocopherol, γ-tocopherol, δ-tocopherol,butylhydroxinon, butyl hydroxyanisole (BHA), butyl hydroxytoluene (BHT),propyl gallate, octyl gallate, dodecyl gallate, tertiarybutylhydroquinone (TBHQ), fumaric acid, malic acid, ascorbic acid(Vitamin C), sodium ascorbate, calcium ascorbate, potassium ascorbate,ascorbyl palmitate, ascorbyl stearate,citric acid, sodium lactate,potassium lactate, calcium lactate, magnesium lactate, anoxomer,erythorbic acid, sodium erythorbate, erythorbin acid, sodium erythorbin,ethoxyquin, glycine, gum guaiac, sodium citrates (monosodium citrate,disodium citrate, trisodium citrate), potassium citrates (monopotassiumcitrate, tripotassium citrate), lecithin, polyphosphate, tartaric acid,sodium tartrates (monosodium tartrate, disodium tartrate), potassiumtartrates (monopotassium tartrate, dipotassium tartrate), sodiumpotassium tartrate, phosphoric acid, sodium phosphates (monosodiumphosphate, disodium phosphate, trisodium phosphate), potassiumphosphates (monopotassium phosphate, dipotassium phosphate, tripotassiumphosphate), calcium disodium ethylene diamine tetra-acetate (Calciumdisodium EDTA), lactic acid, trihydroxy butyrophenone andthiodipropionic acid.

In certain embodiments, the composition of the formulation comprises anantioxidant and both a chelator and a sequestrate. The chelating agentacts to remove trace quantities of metals which might otherwise bind tothe GCC agonist and cause loss of activity, for example throughoxidation. The sequestrate preferably has several hydroxyl and/orcarboxylic acid groups which provide a supply of hydrogen forregeneration of the inactivated antioxidant free radical. Non-limitingexamples of chelating agents include antioxidants, dipotassium edentate,disodium edentate, edetate calcium disodium, edetic acid, fumaric acid,malic acid, maltol, sodium edentate, and trisodium edetate. Non-limitingexamples of sequestrates include citric acid and ascorbic acid.

In some embodiments, the formulation further comprises a filler.Preferably, the filler is present in an amount of from 10% to 85% byweight. Non-limiting examples of suitable materials for use as a fillerinclude starch, lactitol, lactose, an inorganic calcium salt,microcrystalline cellulose, sucrose, and combinations thereof. In someembodiments, the filler comprises microcrystalline cellulose.Preferably, the microcrystalline cellulose has a particle size of lessthan about 100 microns, and most preferably the microcrystallinecellulose has a particle size of about 50 microns.

In some embodiments, the composition optionally includes a bufferingagent such as an inorganic salt compound and an organic alkaline saltcompound. Non-limiting examples of a buffering agent include potassiumbicarbonate, potassium citrate, potassium hydroxide, sodium bicarbonate,sodium citrate, sodium hydroxide, calcium carbonate, dibasic sodiumphosphate, monosodium glutamate, tribasic calcium phosphate,monoethanolamine, diethanolamine, triethanolamine, citric acidmonohydrate, lactic acid, propionic acid, tartaric acid, fumaric acid,malic acid, and monobasic sodium phosphate.

In some embodiments, the composition described herein further comprisesa preservative. Non-limiting examples of a preservative include anantioxidant, dipotassium edentate, disodium edentate, edetate calciumdisodium, edetic acid, fumaric acid, malic acid, maltol, sodiumedentate, and trisodium edentate.

The formulations of the invention are preferably optimized for oraldelivery. However, in some embodiments, the formulations may be preparedin the form of suppositories (e.g., with conventional suppository basessuch as cocoa butter and other glycerides) or retention enemas forrectal delivery. Solid oral dosage forms may optionally be treated withcoating systems (e.g. Opadry® fx film coating system, for exampleOpadry® blue (OY-LS-20921), Opadry® white (YS-2-7063), Opadry® white(YS-1-7040), and black ink (S-1-8 106).

Uses

The present invention provides a method of colonic cleansing byadministering to a subject in need thereof an effective amount of anycompositions of the present invention, for example a GCRA peptide.

The present invention also provides a method of colonic cleansing byadministering to a subject in need thereof an effective amount of anyformulations of the present invention.

The GCRA peptides that can be used in the methods and formulations ofthe invention include a peptide selected from the group designated bySEQ ID NOs: 1-346. Preferably, the GCRA peptide is SEQ ID NO: 1, 9, 55or 56.

This method can be used in cleansing or purging the bowels or colonprior to carrying out a diagnostic, therapeutic or surgical procedure onthe colon, rectum or anus or elsewhere in the abdomen. The diagnostic orsurgical procedure may, for example, be sigmoidoscopy, colonoscopy,radiographic examination, preparation for patients undergoing bowelsurgery, and other medical or diagnostic procedures. It has beenbelieved that profuse, uncontrolled diarrhea was necessary to produceadequate cleansing of the colon. This present invention provides a safeand effective cleansing method for the bowels and colon, without theingestion of large volumes of lavage solutions, without the unpleasant,bitter, and dangerous hypertonic salt solutions, thus providing animproved patients compliance.

“Subject”, as used herein, means an individual. In one aspect, thesubject is a mammal such as a primate, and, in another aspect, thesubject is a human. The term “subject” also includes domesticatedanimals (e.g., cats, dogs, etc.), and livestock (e.g., cattle, horses,pigs, sheep, goats, etc.). The subject may be a human over 50 years old.In some embodiments, the subject is one who is undergoing colonoscopyfor a routinely accepted indication, including, but not limited toroutine screening, polyp or neoplasm history, rectal bleeding, othergastrointestinal bleeding, abdominal pain, unknown diarrhea orconstipation etiology, anemia of unknown etiology, inflammatory boweldisease, abnormal endosonography or evaluation of barium enema results.

The compositions (e.g., GCRA peptides) or the formulations describedherein can be administered with one or more other agents, for example,L-glucose, cholera toxin, osmotic colonic evacuants, cathartic,laxatives and agents for treating chronic constipation. In someembodiments, the compositions (e.g., GCRA peptides) or the formulationsdescribed herein can be administered with L-glucose. The compositions orthe formulations described herein can be administered prior to,concurrently, or after the administration of one or more such agents.

Preferably, the GCRA peptide or its derivative or analog of the presentinvention or any composition/formulation described herein is used incombination with L-glucose, cholera toxin, osmotic colonic evacuants,cathartic, laxatives, agents for treating chronic constipation and/or anosmotic liquid prep. For example, an osmotic liquid prep is SUPREP®Bowel Prep Kit (sodium sulfate potassium sulfate, magnesium sulfate).

The term “cathartic”, as used herein, refers to any composition thatacts as a colonic evacuant for cleansing or purging the bowels or colonprior to sigmoidoscopy, colonoscopy, radiographic examination,preparation for patients undergoing bowel surgery, and other medical ordiagnostic procedures.

The phrase “osmotic colonic evacuant”, as used herein, refers to anycomposition that induces water infusion and retention into theintestinal lumen when the composition is administered to a subject.Compositions of an osmotic colonic evacuant include solids, powders,gels, or liquids. A liquid composition of an osmotic colonic evacuantmay be constituted from a solid, powder, or gel composition using aphysiologically acceptable carrier (e.g., water). A liquid compositionof an osmotic colonic evacuant suitable for administration also may beconstituted from liquid concentrate form using a physiologicallyacceptable carrier (e.g., water) as diluent.

An osmotic colonic evacuant may be a phosphate-based cathartic, forexample, sodium dihydrogen phosphate, disodium hydrogen phosphate,sodium biphosphate, sodium acid pyrophosphate, or mixtures thereof; or asulfate-based cathartic, for example, sodium picosulfate and sodiumsulfate, or mixtures thereof; or magnesium-based cathartic, for example,magnesium citrate, magnesium hydroxide, magnesium sulfate, magnesiumoxide, or mixtures thereof; or magnesium sulfate (Epsom salts).

A stimulant laxative can cause rhythmic muscle contractions in the largeintestines. Exemplary stimulant laxatives and their effective dosesinclude: Aloe, 250-1000 mg.; bisacodyl, about 5-80 mg.; casanthranol, 30to 360 mg.; cascara aromatic fluid extract, 2-24 ml.; cascara sagradabark, 300-4000 mg.; cascada sagrada extract, 300 to 2000 mg.; cascarasagrada fliuid extract, 0.5 to 5 ml.; castor oil, 15-240 ml.; danthron,75-300 mg.; dehydrocholic acid, 250-2000 mg; phenolphthalein, 30-1000mg.; sennosides A and B, 12-200 mg.; and picosulfate, 1-100 mg. Otherlaxatives may include glycerin suppositories, sorbitol, lactulose, andpolyethylene glycol (PEG).

Exemplary agents for treating chronic constipation include, but are notlimited to lubiprostone (Amitiza), prucalopride, SENNA, MIRALAX,LACTULOSE, PEG, or calcium polycarbophil), stool softeners (such asmineral oil or COLACE), bulking agents (such as METAMUCIL or bran),agents such as ZELNORM (also called tegaserod), and anticholinergicmedications such as BENTYL and LEVSIN.

The compositions (e.g., GCRA peptides) or the formulations describedherein can be used in combination with a phosphodiesterase inhibitor.PDE inhibitors are those compounds which slow the degradation of cyclicAMP (cAMP) and/or cyclic GMP (cGMP) by inhibition of thephosphodiesterases, which can lead to a relative increase in theintracellular concentration of c AMP and/or cGMP. Possible PDEinhibitors are primarily those substances which are to be numbered amongthe class consisting of the PDE3 inhibitors, the class consisting of thePDE4 inhibitors and/or the class consisting of the PDES inhibitors, inparticular those substances which can be designated as mixed types ofPDE3/4 inhibitors or as mixed types of PDE3/4/5 inhibitors. By way ofexample, those PDE inhibitors may be mentioned such as are describedand/or claimed in the following patent applications and patents:DE1470341, DE2108438, DE2123328, DE2305339, DE2305575, DE2315801,DE2402908, DE2413935, DE2451417, DE2459090, DE2646469, DE2727481,DE2825048, DE2837161, DE2845220, DE2847621, DE2934747, DE3021792,DE3038166, DE3044568, EP000718, EP0008408, EP0010759, EP0059948,EP0075436, EP0096517, EPO1 12987, EPO1 16948, EP0150937, EP0158380,EP0161632, EP0161918, EP0167121, EP0199127, EP0220044, EP0247725,EP0258191, EP0272910, EP0272914, EP0294647, EP0300726, EP0335386,EP0357788, EP0389282, EP0406958, EP0426180, EP0428302, EP0435811,EP0470805, EP0482208, EP0490823, EP0506194, EP0511865, EP0527117,EP0626939, EP0664289, EP0671389, EP0685474, EP0685475, EP0685479,JP92234389, JP94329652, JP95010875, U.S. Pat. Nos. 4,963,561, 5,141,931,WO9117991, WO9200968, WO9212961, WO9307146, WO9315044, WO9315045,WO9318024, WO9319068, WO9319720, WO9319747, WO9319749, WO9319751,WO9325517, WO9402465, WO9406423, WO9412461, WO9420455, WO9422852,WO9425437, WO9427947, WO9500516, WO9501980, WO9503794, WO9504045,WO9504046, WO9505386, WO9508534, WO9509623, WO9509624, WO9509627,WO9509836, WO9514667, WO9514680, WO9514681, WO9517392, WO9517399,WO9519362, WO9522520, WO9524381, WO9527692, WO9528926, WO9535281,WO9535282, WO9600218, WO9601825, WO9602541, WO9611917, DE3142982, DE1116676, DE2162096, EP0293063, EP0463756, EP0482208, EP0579496, EP0667345U.S. Pat. No. 6,331,543, US20050004222 (including those disclosed informulae I-XIII and paragraphs 37-39, 85-0545 and 557-577) andWO9307124, EP0163965, EP0393500, EP0510562, EP0553174, WO9501338 andWO9603399. PDES inhibitors which may be mentioned by way of example areRX-RA-69, SCH-51866, KT-734, vesnarinone, zaprinast, SKF-96231,ER-21355, BF/GP-385, NM-702 and sildenafil (Viagra®). PDE4 inhibitorswhich may be mentioned by way of example are RO-20-1724, MEM 1414(R1533/R1500; Pharmacia Roche), DENBUFYLLINE, ROLIPRAM, OXAGRELATE,NITRAQUAZONE, Y-590, DH-6471, SKF-94120, MOTAPIZONE, LIXAZINONE,INDOLIDAN, OLPRINONE, ATIZORAM, KS-506-G, DIPAMFYLLINE, BMY-43351,ATIZORAM, AROFYLLINE, FILAMINAST, PDB-093, UCB-29646, CDP-840,SKF-107806, PICLAMILAST, RS-17597, RS-25344-000, SB-207499, TIBENELAST,SB-210667, SB-211572, SB-211600, SB-212066, SB-212179, GW-3600, CDP-840,MOPIDAMOL, ANAGRELIDE, IBUDILAST, AMRINONE, PIMOBENDAN, CILOSTAZOL,QUAZINONE andN-(3,5-dichloropyrid-4-yl)-3-cyclopropylmethoxy4-difluoromethoxybenzamide.PDE3 inhibitors which may be mentioned by way of example are SULMAZOLE,AMPIZONE, CILOSTAMIDE, CARBAZERAN, PIROXIMONE, IMAZODAN, CI-930,SIGUAZODAN, ADIBENDAN, SATERINONE, SKF-95654, SDZ-MKS-492, 349-U-85,EMORADAN, EMD-53998, EMD-57033, NSP-306, NSP-307, REVIZINONE, NM-702,WIN-62582 and WIN-63291, ENOXIMONE and MILRINONE. PDE3/4 inhibitorswhich may be mentioned by way of example are BENAFENTRINE, TREQUINSIN,ORG-30029, ZARDAVERINE, L-686398, SDZ-ISQ-844, ORG-20241, EMD-54622, andTOLAFENTRINE. Other PDE inhibitors include: cilomilast, pentoxifylline,roflumilast, tadalafil (Cialis®), theophylline, and vardenafil(Levitra®), zaprinast (PDES specific). Preferably, the cGMP-specificphosphodiesterase inhibitor is selected from the group consisting ofsulindac sulfone, zaprinast, motapizone, vardenafil, and sildenafil.

Prior to consumption, flavorants and/or sweeteners may be added tocompositions of the present invention to increase its palatability.Optionally, the flavorant can be present in the mixture or solution thatcontains the instant compositions. Alternatively, the flavorant andsweetener can be individually packaged apart from the instantcompositions. The flavorant may include a citrate-based component. Thecitrate-based component may include citric acid, salts, such as sodiumor potassium citrate, derivatives of citrate, such as a citratederivatized with ester functionality, and the like. The flavorant alsomay include natural and/or artificial flavorings, such as natural and/orartificial fruit flavors, to further increase the palatability of thecathartic. Preferred sweeteners include aspartame, sucralose, andacesulfame potassium, among other ingredients. Preferably, the flavorantand sweetener may be combined as a powered mixture. Examples of suchcombinations include the commercially available aspartame-based drinkmixture, such as the CRYSTAL LIGHT®™ powder that is available from KraftFoods, Northfield, Ill. (USA) or the N&A Pink Lemonade FL System SugarFAFT523 that is available from WILD Flavors, Inc., Erlanger, Ky. (USA).Both of these powders include aspartame, citric acid, and fruit flavorsthat result in flavored drinks when the powder is combined with water.Examples of compositions and uses of flavorants and sweeteners inphosphate-based cathartics are described, for example, in ASPARTAME ANDCITRATE FLAVORED PHOSPHATE SALT CATHARTIC, U.S. Published PatentApplication publication no. US20060051428 to Ayala et al.

The GCRA peptides may be in a pharmaceutical composition in unit doseform, together with one or more pharmaceutically acceptable excipients.The term “unit dose form” refers to a single drug delivery entity, e.g.,a tablet, capsule, solution or inhalation formulation. The amount ofpeptide present should be sufficient to have a positive effect whenadministered to a patient (typically, between 10 μg and 3 g). “Positiveeffect” refers to effective cleansing or purging the bowels or colonprior to carrying out a diagnostic, therapeutic or surgical procedure onthe colon, rectum or anus or elsewhere in the abdomen.

The GCRA peptides can be administered alone or in combination with otheragents. Combination methods can be achieved by administering two or moreagents, e.g., a GCRA peptide described herein and another compound, eachof which is formulated and administered separately, or by administeringtwo or more agents in a single formulation. Other combinations are alsoencompassed by combination therapy. For example, two agents can beformulated together and administered in conjunction with a separateformulation containing a third agent. While the two or more agents inthe combination methods can be administered simultaneously, they neednot be. For example, administration of a first agent (or combination ofagents) can precede administration of a second agent (or combination ofagents) by minutes, hours, or days. Thus, the two or more agents can beadministered within minutes of each other or within 1, 2, 3, 6, 9, 12,15, 18, or 24 hours of each other or within 1 or 2 days of each other.In some cases even longer intervals are possible. While in many cases itis desirable that the two or more agents used in a combination therapybe present in within the patient's body at the same time, this need notbe so.

In some embodiments, a GCRA peptide or its derivative or analog of thepresent invention is administered 30 minutes before administering anadditional colon cleansing agent (e.g., L-glucose, cholera toxin,osmotic colonic evacuants, cathartic, laxatives, agents for treatingchronic constipation and/or an osmotic liquid prep).

In some embodiments, a GCRA peptide or its derivative or analog of thepresent invention is administered the evening prior to before thecolonoscopy (e.g., at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18 hours or more before the colonoscopy).

Combination therapy can also include two or more administrations of oneor more of the agents used in the combination. For example, if agent Xand agent Y are used in a combination, one could administer themsequentially in any combination one or more times, e.g., in the orderX-Y-X, X-X-Y, Y-X-Y, Y-Y-X, X-X-Y-Y, etc.

Combination therapy can also include the administration of two or moreagents via different routes or locations. For example, (a) one agent isadministered orally and another agent is administered intravenously or(b) one agent is administered orally and another is administeredlocally. In each case, the agents can either simultaneously orsequentially. Approximated dosages for some of the combination therapyagents described herein are found in the “BNF Recommended Dose” columnof tables on pages 11-17 of WO01/76632 (the data in the tables beingattributed to the March 2000 British National Formulary) and can also befound in other standard formularies and other drug prescribingdirectories. For some drugs, the customary prescribed dose for anindication will vary somewhat from country to country. p In someembodiments, the GCRA peptide or its derivative or analog of the presentinvention or any composition/formulation described herein and theadditional agent (e.g., L-glucose, cholera toxin, osmotic colonicevacuants, cathartic, laxatives, agents for treating chronicconstipation and/or an osmotic liquid prep) are administered orally.

The GCRA peptides, alone or in combination, can be combined with anypharmaceutically acceptable carrier or medium. Thus, they can becombined with materials that do not produce an adverse, allergic orotherwise unwanted reaction when administered to a patient. The carriersor mediums used can include solvents, dispersants, coatings, absorptionpromoting agents, controlled release agents, and one or more inertexcipients (which include starches, polyols, granulating agents,microcrystalline cellulose (e.g. Celphere®, Celphere® beads), diluents,lubricants, binders, disintegrating agents, and the like), etc. Ifdesired, tablet dosages of the disclosed compositions may be coated bystandard aqueous or nonaqueous techniques.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates, and agents for theadjustment of tonicity such as sodium chloride or dextrose. The pH canbe adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorELTM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringeability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound (e.g., a GCRA agonist) in the required amount in an appropriatesolvent with one or a combination of ingredients enumerated above, asrequired, followed by filtered sterilization. Generally, dispersions areprepared by incorporating the active compound into a sterile vehiclethat contains a basic dispersion medium and the required otheringredients from those enumerated above. In the case of sterile powdersfor the preparation of sterile injectable solutions, methods ofpreparation are vacuum drying and freeze-drying that yields a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier, such as mannitol, fructooligosaccharides, polyethylene glycoland other excepients. They can be enclosed in gelatin capsules orcompressed into tablets. For the purpose of oral therapeuticadministration, the active compound can be incorporated with excipientsand used in the form of tablets, troches, or capsules. Oral compositionscan also be prepared using a fluid carrier for use as a mouthwash,wherein the compound in the fluid carrier is applied orally and swishedand expectorated or swallowed. Pharmaceutically compatible bindingagents, and/or adjuvant materials can be included as part of thecomposition. The tablets, pills, capsules, troches and the like cancontain any of the following ingredients, or compounds of a similarnature: a binder such as microcrystalline cellulose, gum tragacanth orgelatin; an excipient such as starch or lactose, a disintegrating agentsuch as alginic acid, Primogel, or corn starch; a lubricant such asmagnesium stearate or Sterotes; a glidant such as colloidal silicondioxide; a sweetening agent such as sucrose or saccharin; or a flavoringagent such as peppermint, methyl salicylate, or orange flavoring.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art. Thecompositions described herein can also be prepared in the form ofsuppositories (e.g., with conventional suppository bases such as cocoabutter and other glycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811, incorporated fully herein by reference.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

Compositions of the present invention may also optionally include othertherapeutic ingredients, anti-caking agents, preservatives, sweeteningagents, colorants, flavors, desiccants, plasticizers, dyes, glidants,anti-adherents, anti-static agents, surfactants (wetting agents),anti-oxidants, film-coating agents, and the like. Any such optionalingredient must be compatible with the compound described herein toinsure the stability of the formulation.

Dosage

Dosage levels of active ingredients in a pharmaceutical composition canalso be varied so as to achieve a transient or sustained concentrationof the compound in a subject, and to result in the desired response. Itis well within the skill of the art to start doses of the compound atlevels lower than required to achieve the desired effect and togradually increase the dosage until the desired effect is achieved. Itwill be understood that the specific dose level for any particularsubject will depend on a variety of factors, including body weight,general health, diet, natural history of disease, route and schedulingof administration, combination with one or more other drugs, andseverity of disease.

An effective dosage of the composition will typically be between about 1μg and about 10 mg per kilogram body weight, preferably between about 10μg to 5 mg of the compound per kilogram body weight. Adjustments indosage will be made using methods that are routine in the art and willbe based upon the particular composition being used and clinicalconsiderations.

The guanylate cyclase receptor agonists used in the methods describedabove may be administered orally, systemically or locally. Dosage formsinclude preparations for inhalation or injection, solutions,suspensions, emulsions, tablets, capsules, topical salves and lotions,transdermal compositions, other known peptide formulations and pegylatedpeptide analogs. Agonists may be administered as either the sole activeagent or in combination with other drugs, e.g., L-glucose, lubiprostone(Amitiza), prucalopride, a laxative, an osmotic colonic evacuant and/oran inhibitor of cGMP-dependent phosphodiesterase. In all cases,additional drugs should be administered at a dosage that istherapeutically effective using the existing art as a guide. Drugs maybe administered in a single composition or sequentially.

Dosage levels of the GCRA peptide for use in methods of this inventiontypically are from about 0.001 mg to about 10,000 mg daily, preferablyfrom about 0.005 mg to about 1,000 mg daily. For example, an effectiveamount of the GCRA peptide for use in methods of this invention is 0.01,0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5,3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55or 60 mg per unit dose. In some preferred embodiments, an effectiveamount of the GCRA peptide for use in methods of this invention is 6.0mg per unit dose. In some embodiments, an effective amount of the GCRApeptide for use in methods of this invention is 1.0-60 mg (e.g., 1.0,1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0,8.5, 9.0, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35,40, 45, 50, 55 or 60 mg) per unit dose, when the GCRA peptide is SP-304(SEQ ID NO: 1), SP-333 (SEQ ID NO: 9) or their derivatives or analogs.In some embodiments, an effective amount of the GCRA peptide for use inmethods of this invention is 0.3-3.0 mg (e.g., 0.3, 0.4, 0.5, 0.6, 0.7,0.8, 0.9, 1.0, 1.5, 2.0, 2.5, or 3.0 mg) per unit dose, when the GCRApeptide is an E. coli ST peptide, linaclotide (SEQ ID NO: 55) or itsderivatives or analogs. In a merely illustrative embodiment, the GCRApeptide is given once or twice one day before the procedure (e.g.,colonoscopy, surgery etc.) and once or twice on the day of the procedurebut before the procedure. In some embodiments a second agent useful forcleansing or purging the bowels or colon prior to carrying out adiagnostic, therapeutic or surgical procedure on the colon, rectum oranus or elsewhere in the abdomen is administered, prior to, concurrentlyor after the administration of the GCRA peptide. Suitable second agentsare described herein (e.g., an osmotic colonic evacuant; L-glucose,lubiprostone (Amitiza), prucalopride; or a cGMP-specificphosphodiesterase inhibitor). In some aspects the second agent isadministered at less than the standard dose for cleansing and purgingthe bowels and colon because the GCR agonist acts synergistically withthe second agent. The dosage of the second agent can be determined by aphysician. For example, the amount of L-glucose is a unit dose of 20g-200 g, preferably a unit dose of 24 g-48 g. On the basis of mg/kgdaily dose, either given in single or divided doses, dosages typicallyrange from about 0.001/75 mg/kg to about 10,000/75 mg/kg, preferablyfrom about 0.005/75 mg/kg to about 1,000/75 mg/kg.

In some embodiments, subdoses can be administered two to six times intotal before the procedure, preferably two to four times before theprocedure, and even more preferably two to three times before theprocedure. Doses can be in immediate release form or sustained releaseform sufficiently effective to obtain the desired control over themedical condition.

The dosage regimen to clean or purge the bowels or colon prior to theprocedure with the combinations and compositions of the presentinvention is selected in accordance with a variety of factors. Thesefactors include, but are not limited to, the type, age, weight, sex,diet, and medical condition of the subject, the route of administration,pharmacological considerations such as the activity, efficacy,pharmacokinetics and toxicology profiles of the particular second agentemployed, whether a drug delivery system is utilized. Thus, the dosageregimen actually employed may vary widely and therefore deviate from thepreferred dosage regimen set forth above.

EXAMPLES Example 1 A Phase 1, Single-Dose, Pilot Clinical Study toAssess the Safety, Tolerability and Efficacy of Oral SP-333 as anAdjunct to SUPREP° Bowel Prep Kit for Cleansing of the Colon Prior toColonoscopic Examination in Adults

TABLE 9 PROTOCOL SYNOPSIS A Phase 1, Single-Dose, Pilot Clinical Studyto Assess the Safety, Tolerability and Efficacy of Oral SP-333 as anAdjunct to SUPREP ® Bowel Prep Kit for Cleansing of Study Title theColon Prior to Colonoscopic Examination in Adults Objectives PrimaryObjective: The primary endpoint of this study is bowel preparationtreatment success as determined by the investigator (bowel preparationwill be rated by the Investigator as “Excellent”, “Good”, “Fair” or“Poor”). Secondary Objectives: Safety and Tolerability of SP-333 whengiven as an adjunct to the SUPREP ® Bowel Prep Kit for cleansing of thecolon prior to colonoscopy. Methodology Open Label, Exploratory SingleDose Study Number of Patients Approximately 10 adult male and femalepatients scheduled to undergo colonoscopy and Population Study DrugSP-333 6.0 mg tablets for oral administration. Treatment Patients willtake one (1) 6 mg tablet of SP-333 the evening before their scheduledcolonoscopy. Study Design This is a phase 1, exploratory, open-label,single-dose, single-center study. Criteria for Primary efficacyendpoint: Efficacy will be assessed on the basis of a binary outcome ofEvaluation: overall bowel preparation treatment success or failure forthe Study Population (defined as Efficacy and Safety those patients whotook study drug and for whom the colonoscopy was initiated and who didnot have an adverse event preventing initiation of the colonoscopyprocedure). Bowel preparation treatment success rate will be described;formal hypothesis testing will not be performed. Exploratory EndpointsThe exploratory endpoints for this study are: Volume of intra-proceduralwater required to improve visualization during the procedure asdetermined by the Investigator; and, Proportion of procedures that reachthe cecum and allow complete visualization of the right colon and cecumas determined by the Investigator Safety will be assessed by descriptivesummary of treatment-emergent adverse events (TEAEs), adverse eventsleading to withdrawal, and serious adverse events (SAEs). Data AnalysisEfficacy analysis Bowel preparation treatment success rate will bedescribed for the study population; formal hypothesis testing will notbe performed. Safety Data The safety analysis data set will include allrandomized subjects who receive at least one dose of study drug.Incidence of treatment emergent adverse events, withdrawals due toadverse events, serious adverse events and concomitant medications willbe listed for each patient and summarized by treatment group.Overall Design of the Study

This clinical study outlined above was designed to assess the effect ofa single oral dose of 6 mg SP-333 when given concurrently with astandard pre-colonoscopy bowel prep—the SUPREP® Bowel Prep Kit(Braintree Laboratories, Braintree, Mass. 02185, USA). The investigatoris a highly experienced gastroenterologist and oversaw all studyactivities and performed all colonoscopies per his usual routine.

SUPREP® Bowel Prep Kit (FDA Approved Split-Dose Regimen)

The SUPREP® Bowel Prep Kit is an osmotic laxative approved and indicatedfor cleansing of the colon as a preparation for colonoscopy in adults.(Note that the Medication Guide that accompanies the SUPREP® Bowel PrepKit was given to the patient as per standard clinical practice.)

Patient Instructions for SUPREP® Bowel Prep Kit and Study DrugAdministration:

Evening before colonoscopy—approximately 6 PM

-   -   1. Take Study Drug approximately 30 minutes before the start of        the first bottle of the bowel prep kit.    -   2. Take a 6 ounce bottle of study preparation and pour the        entire contents into the mixing cup provided. Fill the cup with        cool water to the fill line (16 ounces) and drink the entire cup        of solution.    -   3. Drink two (2) 16 ounce glasses of WATER over the next hour.        Fill the mixing cup with water up to the fill line (16 ounces)        and drink the entire glass.        Morning of colonoscopy

At least 3 hours prior to colonoscopy:

-   -   1. Take the second 6 ounce bottle of study preparation and pour        the entire contents into the mixing cup provided. Fill the cup        with cool water to the fill line (16 ounces) and drink the        entire cup of solution.    -   2. Drink two (2) 16 ounce glasses of WATER over the next hour.        Fill the mixing cup with water up to the fill line (16 ounces)        and drink the entire glass.        -   Patient must complete the solution and additional water at            least 2 hours before colonoscopy.            Introduction            Background

Colonoscopists rate currently available pre-procedure colon cleansingpreparations as “excellent” (i.e., low residual solid and liquid volumeallowing improved visualization) in only 50% of patients. Increasing theproportion of preps that result in optimal visualization, especially ofthe right colon [a segment with traditionally higher miss rates forpolyps and flat lesions], will serve to improve the clinical outcome ofthe colonoscopy.

-   -   Orally administered GC-C agonists have been shown to increase        bowel movement frequency and improve stool consistency in        patients with chronic idiopathic constipation and in patients        with irritable bowel syndrome with constipation. The normal        physiological response to an orally delivered GC-C receptor        agonist is an increase in water transport into the lumen of the        proximal small bowel; the increased water content results in        more frequent bowel movement with a normalized consistency in        these patients.        SP-333 Mechanism of Action

SP-333 is a novel investigational medicinal product that hasdemonstrated potent agonist of the intestinal GC-C receptor in severalin vitro and in vivo pharmacology studies and is now in Phase 2 clinicaldevelopment. It is a synthetic hexadecapeptide designed to mimic theactions of the natriuretic peptide uroguanylin, a member of the guanylinfamily of enteric peptides. Uroguanylin and guanylin are endogenousagonists for the human GC-C receptor that is expressed on the luminalsurface of epithelial cells lining the GI mucosa; this naturallyoccurring GI hormone stimulates the intracellular production of cyclicguanosine monophosphate (cGMP), resulting in activation of the CysticFibrosis Transmembrane Conductance Regulator (CFTR), and therebygenerating water transport into the bowel lumen.

Clinical Experience

Two phase 1 clinical studies with SP-333 have been completed to date anda Phase 2 trial in OIC is in progress. In the Phase I single ascendingoral dose study, conducted in 64 healthy subjects, the following dosesof SP-333 were tested: 0.1, 0.3, 1, 3, 10, 20, 30, and 60 mg. Overall,SP-333 was safe and well tolerated. Diarrhea was the most common adverseevent and was generally dose-related, with 1 mg being the thresholddose. The time to onset of diarrhea was variable but tended to decreasewith increasing doses from 4 to 2 hours or less. The number of subjectswith watery bowel movement rose from none (<3 mg) to 2 of 6 (3, 10 and20 mg) to 4 of 6 (30 mg). The majority of these episodes was mild,occurred once, and was associated with urgency and in some cases mildcramping. There were no SAEs. No clinically meaningful changes inclinical laboratory test results, ECG or vital signs were observed, andno serious adverse events were reported.

In the Phase I multiple ascending dose study, 89 subjects received dailydoses of 0.3, 1, 3, 10, & 30 mg SP-333 for 14 days or 60 mg SP-333 for 7days. The maximum tolerated dose was 30 mg with 60 mg deemed anintolerable dose due to 2 instances of fecal incontinence or soiling inthe 7 patients dosed with 60 mg SP-333. SP-333 was generally safe andwell-tolerated during this study; there were no clinically meaningfulchanges in hematology, clinical chemistry, or urinalysis parameters, andno clinically meaningful changes in vital sign or ECG parameters withSP-333 dosing at any dose level. Most commonly reported as TEAEs(treatment-emergent adverse events) were gastrointestinal disorders(primarily diarrhea and defecation urgency) of mild or moderateintensity. No clinically meaningful changes in clinical laboratory testresults, ECG or vital signs were observed; no subject experienced aserious adverse event (SAE) and no subject withdrew from the study dueto a TEAE.

Phase 1 pharmacokinetic sampling indicated no detectable (limit ofquantitation=1 ng/mL) systemic absorption of SP-333 following oral doses<10 mg; it is therefore highly unlikely that a single oral dose of 6 mgSP-333 will result in any systemic exposure. Detailed informationconcerning the available pharmacology, toxicology, drug metabolism,clinical studies' data and the adverse event profile of SP-333 can befound in the Investigator's Brochure.

Overall, SP-333 is an ideal agent to enhance the effectiveness ofcurrent bowel cleansing regimens by physiologically increasing watercontent in the intestine thereby facilitating better evacuation of bowelcontents prior to colonoscopy.

Rationale and Objectives

Colorectal cancer (CRC) is a leading cause of cancer death. The lifetimerisk of developing CRC in the US approaches 6%, and almost half of thoseaffected will die of the disease. Despite the usefulness of screeningprocedures for its detection, CRC is a major cause of morbidity andmortality. In screening procedures for CRC such as sigmoidoscopy,colonoscopy, and radiography, it is important that the colon bethoroughly purged and cleansed. In particular, it is essential that asmuch fecal matter and fluids as possible be removed from the colon topermit adequate visualization of the intestinal mucosa. Furthermore,should a planned diagnostic procedure become a therapeutic procedure byvirtue of an unexpected pathological finding (e.g., an adenomatous polyprequiring removal), the ease of performance and overall success of thetherapeutic intervention is enhanced when minimal residual fecal matterand fluid are present.

Primary Objective:

The primary objective of this study was to assess the bowel preparationtreatment success of 6 mg SP-333 plus SUPREP® Bowel Prep Kit compared toprevious bowel preparation treatment success experience of theInvestigator using the SUPREP® Bowel Prep Kit used alone.

Secondary Objective:

The secondary objective of this study was to assess the overall safetyand tolerability of 6 mg SP-333 when used as an adjunct to SUPREP® BowelPrep Kit for bowel cleansing prior to colonoscopy.

Exploratory Objectives:

The exploratory objectives of this study were to assess the volume ofintra-procedural water required to improve visualization and theproportion of procedures that reach the cecum and allow completevisualization of the right colon and cecum using 6 mg SP-333 plusSUPREP® Bowel Prep Kit compared to the previous experience of theInvestigator with the SUPREP® Bowel Prep Kit used alone.

Rationale for the Doses Selected

Dose selection for this trial was based on results of the Phase 1studies where a 6 mg oral daily dose of SP-333 was found to result inself-limited diarrhea (watery stools) with mild urgency in healthyvolunteers. No additional AEs of note were observed.

Efficacy Endpoints

Primary Endpoint

The primary endpoint of this study was bowel preparation treatmentsuccess as determined by the investigator (bowel preparation will berated by the Investigator as “Excellent”, “Good”, “Fair” or “Poor”). Asuccessful bowel preparation treatment is defined as bowel cleansinggraded either “Excellent” or “Good” by the investigator (See Table 10).

TABLE 10 Bowel Preparation Treatment Assessment Score Grade Description1 Poor Large amounts of fecal residue, additional cleansing required 2Fair Enough feces or fluid to prevent a completely reliable exam 3 GoodSmall amounts of feces or fluid not interfering with exam 4 Excellent Nomore than small bits of adherent feces/fluidSecondary Endpoints

The secondary endpoints for this study were:

-   -   Frequency of Treatment Emergent Adverse Events    -   Frequency of Serious Adverse Events (SAEs)        Exploratory Endpoints

The exploratory endpoints for this study were:

-   -   Volume of intra-procedural water required to improve        visualization during the procedure as determined by the        Investigator; and,    -   Proportion of procedures that reach the cecum and allow complete        visualization of the right colon and cecum as determined by the        Investigator        Safety Endpoints    -   Frequency of Treatment Emergent Adverse Events    -   Frequency of SAEs    -   Number of patients who were not able to initiate colonoscopy due        to an adverse event        Planned Sample Size and Number of Study Centers

Approximately ten (10) patients requiring routine diagnostic colonoscopywere recruited by the Investigator from his practice.

Duration of Study

Patients were consented for the study at the same time that informedconsent was obtained for the colonoscopy procedure. Each successfullyconsented & recruited patient took a single dose of 6 mg SP-333 in theevening before the colonoscopy—approximately 30 minutes prior toingestion of the first bottle of the SUPREP® Bowel Prep Kit (See Sectionentitled “Patient Instructions for SUPREP® Bowel Prep Kit” above).

Study Population

The study population consisted of male and female outpatients who wereundergoing colonoscopy for a routinely accepted indication.

Inclusion Criteria

Patients were entered into the study only if they meet all of thefollowing criteria:

-   1. Male or female outpatients who are undergoing colonoscopy for a    routinely accepted indication, including (but not limited to routine    screening, polyp or neoplasm history, rectal bleeding, other    gastrointestinal bleeding, abdominal pain, unknown diarrhea or    constipation etiology, anemia of unknown etiology, inflammatory    bowel disease, abnormal endosonography or evaluation of barium enema    results-   2. At least 18 years of age-   3. Females, and of child-bearing potential, and males with female    partners of child-bearing potential are using an acceptable form of    birth control (hormonal birth control, IUD, double-barrier method,    depot contraceptive, abstinent, or vasectomized spouse). Subjects    practicing abstinence must agree to use an acceptable form of birth    control should they become sexually active during the study.    -   Pharmacologic methods of contraception must be stable for at        least one month prior to Visit 1.-   4. Females of child bearing potential must have a negative urine    pregnancy test within 48 hours of the colonoscopy-   5. In the Investigator's judgment, subject is mentally competent to    provide informed consent to participate in the study    Exclusion Criteria    Subjects who meet any of the following criteria were excluded from    the study:-   1. Subjects with known or suspected ileus, severe ulcerative    colitis, gastrointestinal obstruction, gastric retention, bowel    perforation, toxic colitis or megacolon-   2. Subjects who had previous gastrointestinal surgeries (e.g.    colostomy, colectomy, gastric bypass, stomach stapling). Any    questions regarding the significance of a previous gastrointestinal    surgery should be directed to Synergy Pharmaceuticals Inc.-   3. Subjects who, in the opinion of the Principal Investigator, have    an uncontrolled clinically significant pre-existing medical    condition which might put them at undue medical risk for    colonoscopy; examples include, but are not limited to:    -   a. Chronic Child-Pugh Grade B or C liver insufficiency    -   b. renal insufficiency    -   c. Abnormal and clinically significant ECG finding within 3        months prior to scheduled colonoscopy    -   d. History of or current New York Heart Association (NYHA)        Functional Classification grade III or IV congestive heart        failure-   4. Subjects with impaired consciousness that predisposes them to    pulmonary aspiration.-   5. Subjects undergoing colonoscopy for foreign body removal and    decompression.-   6. Subjects who are pregnant or lactating-   7. Subjects of childbearing potential who refuse a pregnancy test.-   8. Subjects who, in the opinion of the Investigator, should not be    included in the study for any reason, including inability to follow    study procedures.-   9. Subjects who have participated in an investigational surgical,    drug, or device study within the past 30 days.-   10. Subjects who withdraw consent before completion of SUPREP® Bowel    Prep Kit    Dietary Restrictions

Subjects may have a light breakfast on the day before colonoscopy,followed by clear liquids until the colonoscopy is completed thefollowing day. Examples of acceptable clear liquids are:

-   -   Water    -   Strained fruit juices (without pulp) including apple, orange,        white grape, or white cranberry    -   Limeade or lemonade    -   Gatorade/Powerade    -   Ginger ale    -   Coffee or tea (do not use any dairy or non-dairy creamer)    -   Chicken broth    -   Gelatin desserts without added fruit or topping

Note: Purple/Red liquids, Milk and Alcoholic beverages are notpermitted.

Variables and Methods of Assessment

Demographics

-   Demographics were assessed at Screening.    Patient Demography-   Patient demography consists of:    -   Age at screening (date of birth)    -   Ethnicity    -   Sex (Gender)-   Descriptive summaries of demographic characteristics were provided.    Efficacy Variables

Efficacy were assessed on the basis of a binary outcome of overall bowelpreparation treatment success or failure for the Study Population(defined as those patients who took study drug and for whom thecolonoscopy was initiated and who did not have an adverse eventpreventing initiation of the colonoscopy procedure). Bowel preparationtreatment success rate was described; formal hypothesis testing was notperformed.

The volume of intra-procedural water required to improve visualization,the proximal insertion depth of the colonoscope and whether or not theright colon and cecum were completely visualized were recorded in theColonoscopy Procedure Record.

Safety Variables

All adverse events were collected from the time of informed consentuntil the end of the study; the SAE reporting period concludedapproximately 30 days after completion of the colonoscopy.

Treatment emergent adverse events were descriptively presented bypreferred term, severity and relationship to treatment.

Procedures by Visit

Visit 1: Screening

-   1. All pre-colonoscopy procedures were performed per the standard of    pre-colonoscopy evaluation as determined important and necessary by    the Investigator.-   2. Proper use of the SUPREP® Bowel Prep Kit was explained per the    routine of Investigator.-   3. Informed consent for colonoscopy was obtained.-   4. Once informed consent was obtained for the colonoscopy:    -   a. Explained SP-333 study and its objectives; allowed subject to        ask any questions he or she may have    -   b. Obtained informed consent for the study-   5. Instructed patient to take study medication in the evening prior    to the colonoscopy—approximately 30 minutes before the start of the    first bottle of the SUPREP® Bowel Prep Kit.    Visit 2 (Day of Colonoscopy)    Visit 2 (Day of Colonoscopy)-   1. When patient arrived for procedure ensured that study drug was    take on the proper timing and assessed patient for continued    suitability for performance of colonoscopy per standard routine.-   2. Ensured that colonoscopy record includes all information as    outlined below.    Study Data Recorded in Colonoscopy Procedure Record-   The following data were collected and recorded in the colonoscopy    procedure record:    -   1) Start time of colonoscopy    -   2) Completion time of colonoscopy    -   3) Volume of water used to improve visualization    -   4) Presence/absence of aphthous ulcerations and characterization    -   5) Presence/absence of ischemic colitis and characterization    -   6) Number of polyps identified and post biopsy characterization    -   7) Number of flat lesions identified and post biopsy        characterization        Drug Accountability

The Investigator, or qualified designee, maintained an accurate recordof the receipt of the study medication as shipped by the Sponsor (ordesignee) and the date received. In addition, accurate records of studydrug dispensed to patients were kept by the Investigator, or qualifieddesignee, including the amount dispensed to each patient, and the datedispensed.

Assessment, Reporting, Recording and Follow Up of Adverse Events

Adverse Event Definition and Reporting

Study patients were monitored and questioned for TEAEs at each studyvisit. An Adverse Event (AE) is any untoward medical occurrenceassociated with the use of a drug in humans, whether or not considereddrug related. An AE can therefore be any unfavorable and unintended sign(including a clinically significant abnormal laboratory finding),symptom, or disease temporally associated with the use of a medicinal(investigational) product. Subjects were queried for any problems theyexperienced during and after preparation by site personnel at allvisits. Colonoscopy and biopsy findings are not considered adverseevents unless considered by the investigator to be related to thepreparation or colonoscopy procedure. Adverse event collection commencedat the time the patient provides informed consent to participate in thestudy and concluded with the completion of the colonoscopy. Subjectswere instructed to promptly report adverse events to the Investigator.The Investigator recorded date/time of report, date/time of onset,description of the adverse event, severity of adverse event, action(s)taken regarding treatment of the event, action(s) taken regarding studyparticipation, duration of adverse event, and the Investigator'sassessment of relationship of adverse event to study treatment. TheInvestigator assessed the severity of each adverse event using thefollowing categories:

Grade Severity Description 1 Mild Barely noticeable, does not influencefunctioning causing no limitations of usual activities 2 Moderate Makesparticipant uncomfortable, influences functioning causing somelimitations of usual activities 3 Severe Severe discomfort, treatmentneeded Severe and undesirable, causing inability to carry out usualactivities 4 Life Immediate risk of death, Life threatening orthreatening disabling (Must be reported as serious adverse event) 5Fatal Causes death of the participant (Must be reported as seriousadverse event)

The Investigator assessed the relationship to study drug for eachadverse event using the following categories:

Categories of Attribution: Description UNRELATED There is no evidence ofany causal relationship. POSSIBLE There is some evidence to suggest acausal relationship (e.g., the event occurred within a reasonable timeafter administration of the trial medication). However, the influence ofother factors may have contributed to the event (e.g., the subject'sclinical condition, other concomitant events). PROBABLE There isevidence to suggest a causal relationship, and the influence of otherfactors is unlikely. DEFINITE There is clear evidence to suggest acausal relationship, and other possible contributing factors can beruled out.Assessment of Adverse Events

-   A Serious Adverse Event (SAE) is any untoward medical occurrence    that results in at least one of the following outcomes:    -   Results in death    -   Is life-threatening    -   Requires inpatient hospitalization or prolongation of existing        hospitalization    -   A persistent or significant incapacity or substantial disruption        of the ability to conduct normal life functions    -   Is a congenital anomaly/birth defect    -   Requires medical or surgical intervention to prevent permanent        impairment or damage        Statistical Methods-   No analyses were carried out other than a descriptive summary of the    primary, secondary, safety and exploratory endpoints of this study.    Results

A total of 9 patients received SP-333 according to the study protocoland completed the study. All patients tolerated the combination ofSP-333 and the standard colonoscopy preparation very well.

The same gastroenterologist performed all colonoscopy on the 9 patients.The gastroenterologist observed that the addition of SP-333 to thestandard colonoscopy cleansing preparation allowed better visualizationof the entire colon and ensured a safe and successful procedure.Subjects who had undergone previous colonoscopies concluded that takingthe SP-333 along with their colon cleansing preparation made thepreparation much easier than their past colon cleansing preparationwithout SP-333.

We claim:
 1. A method of colonic cleansing, comprising administering toa subject in need thereof a unit dose of 0.01 mg to 60 mg of aneffective amount of a guanylate cyclase receptor agonist (GCRA) peptideconsisting essentially of the sequence of SEQ ID NO:
 1. 2. The method ofclaim 1, wherein said peptide is bicyclic consisting essentially of thesequence of SEQ ID NO:
 1. 3. The method of claim 1, further comprisingadministering to said subject an effective amount of an osmotic colonicevacuant.
 4. The method of claim 2, wherein said osmotic colonicevacuant is magnesium citrate.
 5. The method of claim 2, wherein saidosmotic colonic evacuant is a phosphate salt laxative.
 6. The method ofclaim 1, further comprising administering to said subject an effectiveamount of L-glucose, lubiprostone (Amitiza), prucalopride, an agent fortreating chronic constipation, or any combination thereof.
 7. The methodof claim 6, wherein said effective amount of L-glucose is a unit dose of20g-200g.
 8. The method of claim 1, further comprising administering tosaid subject an effective amount of a cGMP-specific phosphodiesteraseinhibitor.
 9. The method of claim 8, wherein said cGMP-specificphosphodiesterase inhibitor is selected from the group consisting ofsulindac sulfone, zaprinast, motapizone, vardenafil, and sildenafil. 10.A method of colonic cleansing, comprising administering to a subject inneed thereof a formulation comprising a mixture of: a. a compositioncomprising an inert carrier coated with GCRA peptides consistingessentially of the sequence of SEP ID NO: 1, wherein said compositioncomprises an enteric coating that releases the peptides at pH 5.0; andb. a composition comprising an inert carrier coated with GCRA peptidesconsisting essentially of the sequence of SEQ ID NO: 1, wherein saidcomposition comprises and enteric coating that releases the peptides atpH 6.0or pH 7.0.